Neuropeptide-2 receptor (Y-2R) agonists and uses thereof

ABSTRACT

Provided herein are neuropeptide-2 receptor agonists of the formula (I):  
                 
as well as pharmaceutically acceptable salts, derivatives and fragments thereof, wherein the substituents are as those disclosed in the specification. These compounds, and the pharmaceutical compositions containing them, are useful for the treatment of diseases such as, for example, obesity and diabetes.

PRIORITY TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/748,071, filed Dec. 7, 2005, and U.S. Provisional Application No.60/855,249, filed Oct. 30, 2006. The entire contents of theabove-identified applications are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to truncated analogs of PYY₃₋₆. The analogs areagonists of neuropeptide-2 receptor and are useful for the treatment ofmetabolic diseases and disorders, such as, for example, obesity, type 2diabetes, metabolic syndrome, insulin resistance and dyslipidemia.

All documents cited herein are hereby expressly incorporated herein byreference.

BACKGROUND OF THE INVENTION

Metabolic diseases and disorders are widely recognized as serious healthproblems for developed countries, having reached epidemic levels in theUnited States. According to recent studies on obesity, for example, morethan 50% of the U.S. population is considered overweight, with more than25% diagnosed as clinically obese and at considerable risk for heartdisease, type 2 diabetes, and certain cancers. This epidemic presents asignificant burden on the health care system as projected obesitytreatment costs of more than $70 billion annually are expected in theU.S. alone. Strategies for treating obesity include reduction of foodintake and enhancing the expenditure of energy.

Neuropeptide Y (NPY), a 36 amino acid peptide neurotransmitter, is amember of the pancreatic polypeptide class ofneurotransmitters/neurohormones which has been shown to be present inboth the periphery and central nervous system. NPY is one of the mostpotent orexigenic agents known and has been shown to play a major rolein the regulation of food intake in animals, including humans.

Six neuropeptide Y receptors (NPY), the Y1-, Y2-, Y3-, Y4, and Y5- andY6-subtypes, have been cloned, which belong to the rhodopsin-likeG-protein-coupled 7-transmembrane spanning receptors (GPCR). The NPY Y2receptor (Y2R) is a 381 amino-acid receptor which inhibits theactivation of adenyl cyclase via G_(i) while displaying low homologywith other known NPY receptors. There is a high degree of conservationbetween rat and human Y2 receptors with 98% amino acid identity.

The Y2R receptor is widely distributed within the central nervous systemin both rodents and humans. In the hypothalamus, Y2 mRNA is localized inthe arcuate nucleus, preoptic nucleus, and dorsomedial nucleus. In thehuman brain, Y2R is the predominant Y receptor subtype. Within thearcuate nucleus, over 80% of the NPY neurons co-express Y2R mRNA.Application of a Y2-selective agonist has been shown to reduce therelease of NPY from hypothalamic slices in vitro, whereas the Y2non-peptide antagonist BIIE0246 increases NPY release. These findingssupport the role of Y2R as a presynaptic autoreceptor that regulates theNPY release and hence may be involved in the regulation of feeding.(Kaga T et al., Peptides 22: 501-506 (2001) and King P J et al., Eur JPharmacol 396: R1-3 (2000)).

Peptide Y₃₋₃₆ (PY₃₋₃₆) is a 34 amino acid linear peptide havingneuropeptide Y2 (NPY2R) agonist activity. It has been demonstrated thatIntra-arcuate (IC) or Intra-peritoneal (IP) injection of PY₃₋₃₆ reducedfeeding in rats and, as a chronic treatment, reduced body weight gain.Intra-venous (IV) infusion (0.8 pmol/kg/min) for 90 min of PY₃₋₃₆reduced food intake in obese and normal human subjects over 24 hours.These finding suggest that the PYY system may be a therapeutic targetfor the treatment of obesity. (Batterham R L et al., Nature 418: 650-654(2002); Batterham R L et al., New Engl J Med 349: 941-948 (2003)).Further, a Cys²-(D)Cys²⁷-cyclized version of PYY, in which residues 5-24were replaced by a methylene-chain of 5 to 8 carbons in length, showedactivation of the intestinal PYY receptor, as evidenced by reducedcurrent across voltage-clamped mucosal preparations of rat jejunum.(Krstenansky, et al. in Peptides, Proceedings of the Twelfth AmericanPeptide Symposium. J. Smith and J. Rivier Editors, ESCOM. Leiden Page136-137).

Further, covalent modification of proteins with poly (ethylene glycol)or poly (ethylene oxide) (both referred to as PEG), was demonstratedwith superoxide dismutase (Somack, R, et al., (1991) Free Rad Res Commun12-13:553-562; U.S. Pat. Nos. 5,283,317 and 5,468,478) and for othertypes of proteins, e.g., cytokines (Saifer, M G P, et al., (1997) PolymPreprints 38:576-577; Sherman, M R, et al., (1997) in J M Harris, etal., (Eds.), Poly(ethylene glycol) Chemistry and BiologicalApplications. ACS Symposium Series 680 (pp. 155-169) Washington, D.C.:American Chemical Society).

In addition, recent data have shown that gastric bypass patients have anearly and exaggerated increase in PYY levels that may be partlyresponsible for the early glycemic control and long term weightmaintenance demonstrating the importance of this peptide in thepathogenesis of metabolic diseases. Other known actions of PYY include:reduced gastric emptying and delayed gastrointestinal transit that isresponsible for improved postprandial glycemic control. Indices ofhyperglycaemia such as HbA_(1C) and fructosamine show a dose-dependentreduction after peripheral administration of PYY₃₋₃₆ in animal models oftype 2 diabetes. Thus, these results indicate that PYY₃₋₃₆, orpharmaceutically related agonists, may offer a long term therapeuticapproach to glycemic and weight control. (Komer et al., J ClinEndocrinol Metabol 90: 359-365 (2005); Chan J L et al., Obesity 14:194-198 (2006); Stratis C et al., Obes Surg 16: 752-758 (2006); Borg C Met al., Br J Surg 93: 210-215 (2006); and Pittner R A et al., Int J Obes28: 963-971 (2004)).

A need exists, however, for novel engineered analogs of PYY having lowermolecular weight, while possessing equal or better potency andselectivity against Y1, Y4 and Y5 receptors, pharmacokinetic propertiesand pharmacological properties. Preferably, a need exists for compoundshaving greater duration of activity than those previously available. Aneed also exists for pegylated analogs of PYY in order to, for example,increase protein half-life and reduce immunogenicity in subjects in needof such agonists.

SUMMARY OF THE INVENTION

In an embodiment of the present invention, provided is a neuropeptide-2receptor agonist of the formula (I):

wherein:

-   X is 4-oxo-6-(1-piperazinyl)-3(4H)-quinazoline-acetic acid (Pqa),-   Y is H, an acyl moiety, a substituted or unsubstituted alkyl, a    substituted or unsubstituted lower alkyl, a substituted or    unsubstituted aryl, a substituted or unsubstituted heteroaryl, a    substituted or unsubstituted alkoxy, a poly(ethylene) glycol moiety,    PEG_(m)-SSA, PEG_(m)-β-SBA, PEG_(m)-SPA or PEG_(m)-BTC,-   Y′ is H, a poly(ethylene) glycol moiety, PEG_(m)-SSA, PEG_(m)-β-SBA,    PEG_(m)-SPA or PEG_(m)-BTC,-   R₁ is Ile, Ala, (D) Ile, N-methyl Ile, Aib, 1-1Aic, 2-2 Aic, Ach or    Acp,-   R₂ is Lys, Ala, (D) Lys, NMelys, Nle or (Lys-Gly),-   R₃ is Arg, Ala, (D)Arg, N-methyl Arg, Phe, 3,4,5-Trifluoro Phe or    2,3,4,5,6-Pentafluoro Phe,-   R₄ is His, Ala, (D)His, N-methyl His, 4-MeOApc, 3-Pal or 4-Pal,-   R₅ is Tyr, Ala, (D) Tyr, N-methyl Tyr, Trp, Tic, Bip, Dip, (1)Nal,    (2)Nal, 3,4,5-Trifluoro Phe or 2,3,4,5,6-Pentafluoro Phe,-   R₆ is Leu, Ala, (D)Leu or N-methyl Leu,-   R₇ is Asn, Ala or (D)Asn,-   R₈ is Leu or Trp,-   R₉ is Val, Ala, (D) Val or N-methyl Val,-   R₁₀ is Thr, Ala or N-methyl Thr,-   R₁₁ is Arg, (D) Arg or N-methyl Arg,-   R₁₂ is Gin or Ala,-   R₁₃ is Arg, (D)Arg or N-methyl Arg,-   R₁₄ is Tyr, (D) Tyr or N-methyl Tyr, modified-Tyr, Phe,    modified-Phe, (1) Nal, (2) Nal, Cha, C-alpha-methyl Tyr, or Trp, and-   PEG_(m) is greater than about 1 KDa,    or a pharmaceutically acceptable salt thereof.

In another embodiment of the present invention, provided is apharmaceutical composition, comprising a therapeutically effectiveamount of a neuropeptide-2 agonist of the formula (I):

wherein:

-   X is 4-oxo-6-(1-piperazinyl)-3(4H)-quinazoline-acetic acid (Pqa),-   Y is H, an acyl moiety, a substituted or unsubstituted alkyl, a    substituted or unsubstituted lower alkyl, a substituted or    unsubstituted aryl, a substituted or unsubstituted heteroaryl, a    substituted or unsubstituted alkoxy, a poly(ethylene) glycol moiety,    PEG_(m)-SSA, PEG_(m)-β-SBA, PEG_(m)-SPA or PEG_(m)-BTC,-   Y′ is H, a poly(ethylene) glycol moiety, PEG_(m)-SSA, PEG_(m)β-SBA,    PEG_(m)-SPA or PEG_(m)-BTC,-   R₁ is Ile, Ala, (D) Ile, N-methyl Ile, Aib, 1-1Aic, 2-2 Aic, Ach or    Acp,-   R₂ is Lys, Ala, (D) Lys, NMelys, Nle or (Lys-Gly),-   R₃ is Arg, Ala, (D)Arg, N-methyl Arg, Phe, 3,4,5-Trifluoro Phe or    2,3,4,5,6-Pentafluoro Phe,-   R₄ is His, Ala, (D)His, N-methyl His, 4-MeOApc, 3-Pal or 4-Pal,-   R₅ is Tyr, Ala, (D) Tyr, N-methyl Tyr, Trp, Tic, Bip, Dip, (1)Nal,    (2)Nal, 3,4,5-TrifluroPhe or 2,3,4,5,6-Pentafluoro Phe,-   R₆ is Leu, Ala, (D)Leu or N-methyl Leu,-   R₇ is Asn, Ala or (D)Asn,-   R₈ is Leu or Trp,-   R₉ is Val, Ala, (D) Val or N-methyl Val,-   R₁₀ is Thr, Ala or N-methyl Thr,-   R₁₁ is Arg, (D) Arg or N-methyl Arg,-   R₁₂ is Gln or Ala,-   R₁₃ is Arg, (D)Arg or N-methyl Arg,-   R₁₄ is Tyr, (D) Tyr or N-methyl Tyr, modified-Tyr, Phe,    modified-Phe, (1) Nal, (2) Nal, Cha, C-alpha-methyl Tyr, or Trp, and-   PEG_(m) is greater than about 1 KDa,    or a pharmaceutically acceptable salt thereof,    and a pharmaceutically acceptable carrier.

In a further embodiment of the present invention, provided is a methodof treating a metabolic disease or disorder, comprising administering toa patient in need of said treatment a therapeutically effective amountof a neuropeptide-2 receptor agonist of the formula (I):

wherein:

-   X is 4-oxo-6-(1-piperazinyl)-3(4H)-quinazoline-acetic acid (Pqa),-   Y is H, an acyl moiety, a substituted or unsubstituted alkyl, a    substituted or unsubstituted lower alkyl, a substituted or    unsubstituted aryl, a substituted or unsubstituted heteroaryl, a    substituted or unsubstituted alkoxy, a poly(ethylene) glycol moiety,    PEG_(m)-SSA, PEG_(m)β-SBA, PEG_(m)-SPA or PEG_(m)-BTC,-   Y′ is H, a poly(ethylene) glycol moiety, PEG_(m)-SSA, PEG_(m)β-SBA,    PEG_(m)-SPA or PEG_(m)-BTC,-   R₁ is Ile, Ala, (D) Ile, N-methyl Ile, Aib, 1-1 Aic, 2-2 Aic, Ach or    Acp,-   R₂ is Lys, Ala, (D) Lys, NMelys, Nle or (Lys-Gly),-   R₃ is Arg, Ala, (D)Arg, N-methyl Arg, Phe, 3,4,5-Trifluoro Phe or    2,3,4,5,6-Pentafluoro Phe,-   R₄ is His, Ala, (D)His, N-methyl His, 4-MeOApc, 3-Pal or 4-Pal,-   R₅ is Tyr, Ala, (D) Tyr, N-methyl Tyr, Trp, Tic, Bip, Dip, (1)Nal,    (2)Nal, 3,4,5-TrifluroPhe or 2,3,4,5,6-Pentafluoro Phe,-   R₆ is Leu, Ala, (D)Leu or N-methyl Leu,-   R₇ is Asn, Ala or (D)Asn,-   R₈ is Leu or Trp,-   R₉ is Val, Ala, (D) Val or N-methyl Val,-   R₁₀ is Thr, Ala or N-methyl Thr,-   R₁₁ is Arg, (D) Arg or N-methyl Arg,-   R₁₂ is Gln or Ala,-   R₁₃ is Arg, (D)Arg or N-methyl Arg,-   R₁₄ is Tyr, (D) Tyr or N-methyl Tyr, modified-Tyr, Phe,    modified-Phe, (1) Nal, (2) Nal, Cha, C-alpha-methyl Tyr, or Trp, and-   PEG_(m) is greater than about 1 KDa,    or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an HPLC chromatogram of a reaction mixture containing acompound (example 34) of the present invention.

FIG. 2 shows an HPLC chromatogram of a purified compound (example 34) ofthe present invention.

FIG. 3 shows a MALDI-TOF spectrum of a compound (example 34) of thepresent invention.

FIG. 4 shows an HPLC chromatogram of a reaction mixture of anothercompound (example 35) of the present invention.

FIG. 5 shows an HPLC chromatogram of a purified a compound (example 35)of the present invention.

FIG. 6 shows a MALDI-TOF spectrum of a compound (example 35) of thepresent invention.

FIG. 7 shows an HPLC chromatogram of a reaction mixture of a compound(example 36) of the present invention.

FIG. 8 shows an HPLC chromatogram of a purified compound (example 36) ofthe present invention.

FIG. 9 shows a MALDI-TOF spectrum of yet another compound (example 36)of the present invention.

FIG. 10 shows an HPLC chromatogram of a reaction mixture of a compound(example 37) of the present invention.

FIG. 11 shows an HPLC chromatogram of a purified compound (example 37)of the present invention.

FIG. 12 shows a MALDI-TOF spectrum of a compound (example 37) of thepresent invention.

FIG. 13 shows an HPLC chromatogram of a reaction mixture of anothercompound (example 38) of the present invention.

FIG. 14 shows HPLC chromatogram of a purified compound (example 38) ofthe present invention.

FIG. 15 shows a MALDI-TOF spectrum of a compound (example 38) of thepresent invention.

FIG. 16 shows an HPLC chromatogram of a reaction mixture of a compound(example 39) of the present invention.

FIG. 17 shows an HPLC chromatogram of a purified compound (example 39)of the present invention.

FIG. 18 shows a MALDI-TOF spectrum of yet another compound (example 39)of the present invention.

FIG. 19 shows an HPLC chromatogram of a reaction mixture containing acompound (example 41) of the present invention before deprotection.

FIG. 20 shows an HPLC chromatogram of a deprotected reaction mixturecontaining a compound (example 41) of the present invention.

FIG. 21 shows an HPLC chromatogram of a purified compound (example 41)of the present invention.

FIG. 22 shows a MALDI-TOF spectrum of a compound (example 41) of thepresent invention.

FIG. 23 shows the effect of sub-chronic dosing of a compound (example41) on body weight in male diet-induced obese (DIO) rats.

FIG. 24 shows the acute effect of a compound (example 41) on an oralglucose tolerance test (OGTT) in female db/db mice.

FIG. 25 shows the effect of sub-chronic dosing of a compound (example41) on basal blood glucose (A) and oral glucose tolerance test (B) infemale db/db mice.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the invention are advantageous because, for example,they are truncated versions of the PY₃₋₃₆ The shorter peptides, forexample, not only facilitate easier synthesis and purification of thecompounds, but also improve and reduce manufacturing procedures andexpenses. Moreover, the compounds of the invention will interactpreferably with Y2-receptors and not with homologous receptors such asNPY Y1, Y4 and Y5. Unwanted agonist or antagonist side reactions are,thereby, minimized.

The compounds of the invention are preferably useful for treatingmetabolic diseases and disorders. Such metabolic diseases and disordersinclude, for example, obesity, diabetes, preferably type 2 diabetes,metabolic syndrome (also known as Syndrome X), insulin resistance,dyslipidemia, impaired fasting glucose and impaired glucose tolerance.

It is to be understood that the invention is not limited to theparticular embodiments of the invention described herein, as variationsof the particular embodiments may be made and still fall within thescope of the appended claims. It is also to be understood that theterminology employed is for the purpose of describing particularembodiments, and is not intended to be limiting. Instead, the scope ofthe present invention will be established by the appended claims.

Although any methods, devices and materials similar or equivalent tothose described herein can be used in the practice or testing of theinvention, the preferred methods, devices and materials are nowdescribed.

All peptide sequences mentioned herein are written according to theusual convention whereby by the N-terminal amino acid is on the left andthe C-terminal amino acid is on the right, unless noted otherwise. Ashort line between two amino acid residues indicates a peptide bond.Where the amino acid has isomeric forms, it is the L form of the aminoacid that is represented unless otherwise expressly indicated. Forconvenience in describing this invention, the conventional andnonconventional abbreviations for the various amino acids are used.These abbreviations are familiar to those skilled in the art, but forclarity are listed below:

-   Asp=D=Aspartic Acid; Ala=A=Alanine; Arg=R=Arginine;    Asn=N=Asparagine; Gly=G=Glycine; Glu=E=Glutamic Acid;    Gln=Q=Glutamine; His=H=Histidine; Ile=I=Isoleucine; Leu=L=Leucine;    Lys=K=Lysine; Met=M=Methionine; Phe=F=Phenylalanine; Pro=P=Proline;    Ser=S=Serine; Thr=T=Threonine; Trp=W=Tryptophan; Tyr=Y=Tyrosine;    Cys=C=Cysteine; and Val=V=Valine.

Also for convenience, the following abbreviations or symbols are used torepresent the moieties, reagents and the like used in this invention:

-   Aib alpha-aminoisobutyric acid;-   1-1-Aic 1-aminoindane-1-carboxylic acid;-   2-2-Aic 2-aminoindane-2-carboxylic acid;-   Ach alpha-aminocyclohexane-carboxylic acid;-   Acp alpha-aminocyclopentane-carboxylic acid;-   Tic alpha-amino-1,2,3,4, tetrahydroisoquinoline-3-carboxylic acid;-   3-Pal alpha-amino-3-pyridylalanine-carboxylic acid;-   4-Pal alpha-amino-4-pyridylalanine-carboxylic acid;-   4-MeO-Apc 1-amino-4-(4-methoxyphenyl)-cyclohexane-1-carboxylic acid;-   Bip 4-phenyl-phenylalanine-caroxylic acid;-   Dip 3,3-diphenylalanine-carboxylic acid;-   Pqa 4-oxo-6-(1-piperazinyl)-3(4H)-quinazoline-acetic acid (CAS    889958-08-1);-   3,4,5, F3-Phe 3,4,5-Trifluoro phenylalanine;-   2,3,4,5,6, F5-Phe 2,3,4,5,6-Pentafluoro phenylalanine;-   Cha Cyclohexyl Alanine;-   Modified-Tyr any modification on tyrosine such as, for example,    methyl-tyrosine, 3-iodo-tyrosine, 3,5-difluoro-tyrosine,    2,6-di-fluoro tyrosine and 2,6-dimethyl-tyrosine;-   Modified-Phe any modification on phenylalanine such as, for example,    4-methoxy-phenylalanine, 4-amino-phenylalanine,    4-fluoro-phenylalanine, 4-hydroxymethyl-phenylalanine,    4-trifluoromethyl-phenylalanine, 3-fluoro-phenylalanine,    2,3,4,5-pentafluoro-phenylalanine and 3,4-dichloro phenylalanine;-   Nle Nor-leucine;-   (1) Nal 1-Naphtyl Alanine;-   (2) Nal 2-Naphtyl Alanine;-   Fmoc 9-Fluorenylmethyloxycarbonyl;-   Mtt 4-Methyltrityl;-   2Pip 2-Phenylisopropyl ester;-   Pmc 2,2,5,7,8-Pentamethylchroman-6-sulfonyl;-   CH₂Cl₂ Methylene chloride;-   A2O Acetic anhydride;-   CH₃CN Acetonitrile;-   DMAc Dimethylacetamide;-   DMF Dimethylformamide;-   DIPEA N,N-Diisopropylethylamine;-   TFA Trifluoroacetic acid;-   HOBT N-Hydroxybenzotriazole;-   DIC N,N′-Diisopropylcarbodiimide;-   BOP Benzotriazol-1-yloxy-tris-(dimethylamino)    phosphonium-Hexafluorophosphate;-   HBTU    2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium-Hexafluorophosphate;-   NMP 1-methyl 2-Pyrolidenone;-   SSA succinimidyl succinamide;-   β-SBA succinimidyl beta-butanoic acid;-   SPA succinimidyl proprionic acid;-   BTC benzotriazole carbonate;-   MALDI-TOF Matrix assisted laser desorption ionization-time of    flight;-   FAB-MS Fast atom bombardment mass spectrometry;-   ES-MS Electro spray mass spectrometry;-   PEG_(m)-SSA PEG_(m)-CH₂CH₂NHCOCH₂CH₂CO—;-   PEG_(m)β-SBA PEG_(m)-CH(CH₃)CH₂CO—;-   PEG_(m)-SPA PEG_(m)-CH₂CH₂CO—;-   PEG_(m)-BTC PEG_(m)-CO—; and-   PEG_(m) is greater than about 1 KDa.

In a preferred embodiment, PEG_(m) is about 1 to about 60 KDa. Morepreferably, PEG_(m) is about 20 to about 40 KDa; most preferably, about30 KDa.

As used herein, the term “alkyl” means a branched or unbranched, cyclicor acyclic, saturated or unsaturated hydrocarbyl radical which may besubstituted or unsubstituted. Where cyclic, the alkyl group ispreferably C₃ to C₁₂, more preferably C₅ to C₁₀, more preferably C₅ toC₇. Where acyclic, the alkyl group is preferably C₁ to C₁₀, morepreferably C₁ to C₆, more preferably methyl, ethyl, propyl (n-propyl orisopropyl), butyl (n-butyl, isobutyl or tertiary-butyl) or pentyl(including n-pentyl and isopentyl), more preferably methyl. It will beappreciated therefore that the term “alkyl” as used herein includesalkyl (branched or unbranched), substituted alkyl (branched orunbranched), substituted alkynyl (branched or unbranched), cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,cycloalkynyl and substituted cycloalkynyl.

As used herein, the term “lower alkyl” means a branched or unbranched,cyclic or acyclic, saturated or unsaturated hydrocarbyl radical whereinsaid cyclic lower alkyl group is C₅, C₆ or C₇, and wherein said acycliclower alkyl group is C₁, C₂, C₃ or C₄, and is preferably selected frommethyl, ethyl, propyl (n-propyl or isopropyl) or butyl (n-butyl,isobutyl or tertiary-butyl). It will be appreciated therefore that theterm “lower alkyl” as used herein includes lower alkyl (branched orunbranched), lower alkenyl (branched or unbranched), lower alkynyl(branched or unbranched), cycloloweralkyl and cycloloweralkenyl.

As used herein, the term “acyl” means an optionally substituted alkyl,cycloalkyl, heterocyclic, aryl or heteroaryl group bound via a carbonylgroup and includes groups such as acetyl, propionyl, benzoyl,3-pyridinylcarbonyl, 2-morpholinocarbonyl, 4-hydroxybutanoyl,4-fluorobenzoyl, 2-naphthoyl, 2-phenylacetyl, 2-methoxyacetyl and thelike.

As used herein, the term “aryl” means a substituted or unsubstitutedcarbocyclic aromatic group, such as phenyl or naphthyl.

The term “heteroaryl”, alone or in combination with other groups, meansa monocyclic or bicyclic radical of 5 to 12 ring atoms having at leastone aromatic ring containing one, two, or three ring heteroatomsselected from N, O, and S, the remaining ring atoms being C, with theunderstanding that the attachment point of the heteroaryl radical willbe on an aromatic ring. One or two ring carbon atoms of the heteroarylgroup may be replaced with a carbonyl group.

The alkyl, aryl and heteroaryl groups may be substituted orunsubstituted. Where substituted, there will generally be 1 to 3substituents present, preferably 1 substituent. Substituents mayinclude: carbon-containing groups such as alkyl, aryl, arylalkyl (e.g.substituted and unsubstituted phenyl, substituted and unsubstitutedbenzyl); halogen atoms and halogen-containing groups such as haloalkyl(e.g. trifluoromethyl); oxygen-containing groups such as alcohols (e.g.hydroxyl, hydroxyalkyl, aryl(hydroxyl)alkyl), alkoxy, aryloxy,alkoxyalkyl, aryloxyalkyl, acyl, acids (e.g. carboxy, carboxyalkyl),acid derivatives such as esters (e.g. alkoxycarbonyl,alkoxycarbonylalkyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl), amides(e.g. aminocarbonyl, mono- or di-alkylaminocarbonyl, aminocarbonylalkyl,mono- or di-alkylaminocarbonylalkyl, arylaminocarbonyl), carbamates(e.g. alkoxycarbonylamino, arloxycarbonylamino, aminocarbonyloxy, mono-or di-alkylaminocarbonyloxy, arylminocarbonloxy) and ureas (e.g. mono-or di-alkylaminocarbonylamino or arylaminocarbonylamino);nitrogen-containing groups such as amines (e.g. amino, mono- ordi-alkylamino, aminoalkyl, mono- or di-alkylaminoalkyl), azides,nitriles (e.g. cyano, cyanoalkyl), nitro; sulfur-containing groups suchas thiols, thioethers, sulfoxides and sulfones (e.g. alkylthio,alkylsulfinyl, alkylsulfonyl, alkylthioalkyl, alkylsulfinylalkyl,alkylsulfonylalkyl, arylthio, arysulfinyl, arysulfonyl, arythioalkyl,arylsulfinylalkyl, arylsulfonylalkyl); and heterocyclic groupscontaining one or more, preferably one, heteroatom, (e.g. thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl,oxazolyl, oxadiazolyl, thiadiazolyl, aziridinyl, azetidinyl,pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl,tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl,piperidyl, hexahydroazepinyl, peperazinyl, morpholinyl, thianaphthyl,benzofuranyl, isobenzofuranyl, indolyl, oxyindolyl, isoindolyl,indazolyl, indolinyl, 7-azaindolyl, benzopyranyl, coumarinyl,isocoumarinyl, quinolinyl, isoquinolinyl, naphthridinyl, cinnolinyl,quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxalinyl, chromenyl,chromanyl, isochromanyl, phthalazinyl and carbolinyl).

The lower alkyl groups may be substituted or unsubstituted, preferablyunsubstituted. Where substituted, there will generally be 1 to 3substitutents present, preferably 1 substituent. Substituents includethe substituent groups listed above other than alkyl, aryl andarylalkyl.

As used herein, the term “alkoxy” means alkyl-O— and “alkanoyl” meansalkyl-CO—. Alkoxy substituent groups or alkoxy-containing substituentgroups may be substituted by one or more alkyl groups.

As used herein, the term “halogen” means a fluorine, chlorine, bromineor iodine radical, preferably a fluorine, chlorine or bromine radical,and more preferably a fluorine or chlorine radical.

“Pharmaceutically acceptable salt” refers to conventional acid-additionsalts or base-addition salts that retain the biological effectivenessand properties of the compounds of formula I and are formed fromsuitable non-toxic organic or inorganic acids or organic or inorganicbases. Sample acid-addition salts include those derived from inorganicacids such as hydrochloric acid, hydrobromic acid, hydroiodic acid,sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and thosederived from organic acids such as acetic acid, p-toluenesulfonic acid,salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citricacid, malic acid, lactic acid, fumaric acid, and the like. Samplebase-addition salts include those derived from ammonium, potassium,sodium and, quaternary ammonium hydroxides, such as for example,tetramethylammonium hydroxide. The chemical modification of apharmaceutical compound (i.e. drug) into a salt is a well knowntechnique which is used in attempting to improve properties involvingphysical or chemical stability, e.g., hygroscopicity, flowability orsolubility of compounds. See, e.g., H. Ansel et. al., PharmaceuticalDosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and1456-1457.

“Pharmaceutically acceptable ester” refers to a conventionallyesterified compound of formula I having a carboxyl group, which estersmay retain the biological effectiveness and properties of the compoundsof formula I and are cleaved in vivo (in the organism) to thecorresponding active carboxylic acid. Examples of ester groups which arecleaved (in this case hydrolyzed) in vivo to the correspondingcarboxylic acids are those in which the cleaved hydrogen is replacedwith lower alkyl which is optionally substituted, e.g., withheterocycle, cycloalkyl, etc. Examples of substituted lower alkyl estersare those in which -lower alkyl is substituted with pyrrolidine,piperidine, morpholine, N-methylpiperazine, etc. The group which iscleaved in vivo may be, for example, ethyl, morpholino ethyl, anddiethylamino ethyl. In connection with the present invention, —CONH₂ isalso considered an ester, as the —NH₂ is cleaved in vivo and replacedwith a hydroxy group, to form the corresponding carboxylic acid.

Further information concerning examples of and the use of esters for thedelivery of pharmaceutical compounds is available in Design of Prodrugs.Bundgaard H. ed. (Elsevier, 1985). See also, H. Ansel et. al.,Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) atpp. 108-109; Krogsgaard-Larsen, et. al., Textbook of Drug Design andDevelopment (2d Ed. 1996) at pp. 152-191

The present representative compounds may be readily synthesized by anyknown conventional procedure for the formation of a peptide linkagebetween amino acids. Such conventional procedures include, for example,any solution phase procedure permitting a condensation between the freealpha amino group of an amino acid or residue thereof having itscarboxyl group and other reactive groups protected and the free primarycarboxyl group of another amino acid or residue thereof having its aminogroup or other reactive groups protected.

Such conventional procedures for synthesizing the novel compounds of thepresent invention include for example any solid phase peptide synthesismethod. In such a method the synthesis of the novel compounds can becarried out by sequentially incorporating the desired amino acidresidues one at a time into the growing peptide chain according to thegeneral principles of solid phase methods. Such methods are disclosedin, for example, Merrifield, R. B., J. Amer. Chem. Soc. 85, 2149-2154(1963); Barany et al., The Peptides, Analysis, Synthesis and Biology,Vol. 2, Gross, E. and Meienhofer, J., Eds. Academic Press 1-284 (1980),which are incorporated herein by reference.

Common to chemical syntheses of peptides is the protection of reactiveside chain groups of the various amino acid moieties with suitableprotecting groups, which will prevent a chemical reaction from occurringat that site until the protecting group is ultimately removed. Usuallyalso common is the protection of the alpha amino group on an amino acidor fragment while that entity reacts at the carboxyl group, followed bythe selective removal of the alpha amino protecting group at allow asubsequent reaction to take place at that site. While specificprotecting groups have been disclosed in regard to the solid phasesynthesis method, it should be noted that each amino acid can beprotected by a protective group conventionally used for the respectiveamino acid in solution phase synthesis.

Alpha amino groups may be protected by a suitable protecting groupselected from aromatic urethane-type protecting groups, such asallyloxycarbonyl, benzyloxycarbonyl (Z) and substitutedbenzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,p-biphenyl-isopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) andp-methoxybenzyloxycarbonyl (Moz); aliphatic urethane-type protectinggroups, such as t-butyloxycarbonyl (Boc), diisopropylmethyloxycarbonyl,isopropyloxycarbonyl, and allyloxycarbonyl. Herein, Fmoc is mostpreferred for alpha amino protection.

Guanidino groups may be protected by a suitable protecting groupselected from nitro, p-toluenesulfonyl (Tos), (Z,)pentamethylchromanesulfonyl (Pmc),4-Methoxy-2,3,6,-trimethylbenzzenesulfonyl (Mtr), (Pmc), and (Mtr) aremost preferred for arginine (Arg).

The ε-amino groups may be protected by a suitable protecting groupselected from 2-chloro benzyloxycarbonyl (2-Cl-Z), 2-Bromobenztloxycarbonyl (2-Br-Z)- and t-butyloxycarbonyl (Boc). Boc is themost preferred for (Lys).

Hydroxyl groups (OH) may be protected by a suitable protecting groupselected from benzyl (Bzl), 2,6 dichlorobenztl (2,6 diCl-Bzl), and tert.Butyl (t-Bu), (tBu) is most preferred for (Tyr), (Ser) and (Thr).

The β- and γ-amide groups may be protected by a suitable protectinggroup selected from 4-methyltrityl (Mtt), 2,4,6-trimethoxybenzyl (Tmob),4, 4-DimethoxyditylBis-(4-methoxyphenyl)-methyl (Dod) and Trityl (Trt).Trt is the most preferred for (Asn) and (Gln).

The indole group may be protected by a suitable protecting groupselected from formyl (For), Mesityl-2-sulfonyl (Mts) andt-butyloxycarbonyl (Boc). Boc is the most preferred for (Trp).

The imidazol group may be protected by a suitable protecting groupselected from Benzyl (Bzl), -t-butyloxycarbonyl (Boc), and Trityl (Trt).Trt is the most preferred for (His).

The synthesis of the amino acid Pqa is described by J. Hutchinson et. al(J. Med. Chem. 1996, 39, 4583-4591). The Fmoc-Pqa derivative waspurchased from NeoMPS, Inc. (San Diego Calif.)

All solvents, isopropanol (iPrOH), methylene chloride (CH₂Cl₂),dimethylformamide (DMF) and N-methylpyrrolinone (NMP) were purchasedfrom Fisher or Burdick & Jackson and were used without additionaldistillation. Trifluoroacetic acid was purchased from Halocarbon orFluka and used without further purification.

Diisopropylcarbodiimide (DIC) and diisopropylethylamine (DIPEA) waspurchased from Fluka or Aldrich and used without further purification.Hydroxybenzotriazole (HOBT) dimethylsulfide (DMS) and 1,2-ethanedithiol(EDT) were purchased from Sigma Chemical Co. and used without furtherpurification. Protected amino acids were generally of the Lconfiguration and were obtained commercially from Bachem, or Neosystem.Purity of these reagents was confirmed by thin layer chromatography, NMRand melting point prior to use. Benzhydrylamine resin (BHA) was acopolymer of styrene-1% divinylbenzene (100-200 or 200-400 mesh)obtained from Bachem or Advanced Chemtech. Total nitrogen content ofthese resins were generally between 0.3-1.2 meq/g.

In a preferred embodiment, peptides were prepared using solid phasesynthesis by the method generally described by Merrifield, (J. Amer.Chem. Soc., 85, 2149 (1963)), although other equivalent chemicalsynthesis known in the art could be used as previously mentioned. Solidphase synthesis is commenced from the C-terminal end of the peptide bycoupling a protected alpha-amino acid to a suitable resin. Such astarting material can be prepared by attaching an alpha-amino-protectedamino acid by an ester linkage to a p-benzyloxybenzyl alcohol (Wang)resin, or by an amide bond between an Fmoc-Linker, such asp-((R,S)-α-(1-(9H-fluoren-9-yl)-methoxyformamido)-2,4-dimethyloxybenzyl)-phenoxyaceticacid (Rink linker) to a benzhydrylamine (BHA) resin. Preparation of thehydroxymethyl resin is well known in the art. Fmoc-Linker-BHA resinsupports are commercially available and generally used when the desiredpeptide being synthesized has an unsubstituted amide at the C-terminus.

Typically, the amino acids or mimetic are coupled onto theFmoc-Linker-BHA resin using the Fmoc protected form of amino acid ormimetic, with 2-5 equivalents of amino acid and a suitable couplingreagent. After couplings, the resin may be washed and dried undervacuum. Loading of the amino acid onto the resin may be determined byamino acid analysis of an aliquot of Fmoc-amino acid resin or bydetermination of Fmoc groups by UV analysis. Any unreacted amino groupsmay be capped by reacting the resin with acetic anhydride anddiispropylethylamine in methylene chloride.

The resins are carried through several repetitive cycles to add aminoacids sequentially. The alpha amino Fmoc protecting groups are removedunder basic conditions. Piperidine, piperazine or morpholine (20-40%v/v) in DMF may be used for this purpose. Preferably 40% piperidine inDMF is utilized.

Following the removal of the alpha amino protecting group, thesubsequent protected amino acids are coupled stepwise in the desiredorder to obtain an intermediate, protected peptide-resin. The activatingreagents used for coupling of the amino acids in the solid phasesynthesis of the peptides are well known in the art. For example,appropriate reagents for such syntheses arebenzotriazol-1-yloxy-tri-(dimethylamino) phosphonium hexafluorophosphate(BOP), Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP)2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), and diisopropylcarbodiimide (DIC). Preferred here are HBTU andDIC. Other activating agents are described by Barany and Merrifield (inThe Peptides, Vol. 2, J. Meienhofer, ed., Academic Press, 1979, pp1-284) and may be utilized. Various reagents such as 1hydroxybenzotriazole (HOBT), N-hydroxysuccinimide (HOSU) and3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBT) may be added tothe coupling mixtures in order to optimize the synthetic cycles.Preferred here is HOBT.

For preparation of N-terminal acetyl derivatives, acetylation wascarried out by treating the resin bound peptide with 20% aceticanhydride in DMF with 5% DIEA. For other N-terminal acylations,acylation was carried out using the corresponding carboxylic acidactivated in-situ with DIC/HOBt for 30 minutes.

The protocol for a typical synthetic cycle is as follows: Protocol 1Step Reagent Time 1 DMF 2 × 30 sec. 2 20% piperidine/DMF 1 min. 3 20%piperidine/DMF 15 min. 4 DMF 2 × 30 sec. 5 iPrOH 2 × 30 sec. 6 DMF 3 ×30 sec. 7 Coupling 60 min-18 hours. 8 DMF 2 × 30 sec. 9 iPrOH 1 × 30sec. 10 DMF 1 × 30 sec. 11 CH₂Cl₂ 2 × 30 sec.

Solvents for all washings and couplings were measured to volumes of10-20 ml/g resins. Coupling reactions throughout the synthesis weremonitored by the Kaiser Ninhydrin test to determine extent of completion(Kaiser et at. Anal. Biochem. 34, 595-598 (1970)). Slow reactionkinetics was observed for Fmoc-Arg (Pmc) and for couplings to secondaryamines by sterically hindered acids. Any incomplete coupling reactionswere either recoupled with freshly prepared activated amino acid orcapped by treating the peptide resin with acetic anhydride as describedabove. The fully assembled peptide-resins were dried in vacuum forseveral hours.

For most compounds, the blocking groups were removed and the peptidecleaved from the resin. For example, the peptide-resins were treatedwith 100 μL ethanedithiol, 100 μl dimethylsulfide, 300 μL anisole, and9.5 mL trifluoroacetic acid, per gram of resin, at room temperature for180 min. Or alternately the peptide-resins were treated with 1.0 mLtriisopropyl silane and 9.5 mL trifluoroacetic acid, per gram of resin,at room temperature for 180 min. The resin was filtered off and thefiltrates were precipitated in chilled ethyl ether. The precipitateswere centrifuged and the ether layer was decanted. The residue waswashed with two or three volumes of Et₂O and recentrifuged. The crudeproducts were dried under vacuum.

Purification of the crude peptides was preferably performed on ShimadzuLC-8A system by high performance liquid chromatography (HPLC) on areverse phase C-18 Column (50×250 mm. 300 A, 10-15 μm). The peptideswere injected to the columns in a minimum volume of either 0.1 AcOH/H₂Oor CH3CH/H₂O. Gradient elution was generally started at 2% B buffer,2%-70% B over 70 minutes, (buffer A: 0.1% TFA/H₂O, buffer B: 0.1%TFA/CH₃CN) at a flow rate of 50 ml/min. UV detection was made at 220/280nm. The fractions containing the products were separated and theirpurity was judged on Shimadzu LC-10AT analytical system using reversephase Ace C18 column (4.6×50 mm) at a flow rate of 2 ml/min., gradient(2-70%) over 10 min. (buffer A: 0.1% TFA/H₂O, buffer B: 0.1%TFA/CH₃CN)). Fractions judged to be of high purity were pooled andlyophilized.

Purity of the final products was checked by analytical HPLC on areversed phase column as stated above. Purity of all products was judgedto be approximately 95-99%. All final products were also subjected tofast atom bombardment mass spectrometry (FAB-MS) or electrospray massspectrometry (ES-MS). All products yielded the expected parent M+H ionswithin acceptable limits.

The compounds of the present invention can be provided in the form ofpharmaceutically acceptable salts. Examples of preferred salts are thoseformed with pharmaceutically acceptable organic acids, e.g., acetic,lactic, maleic, citric, malic, ascorbic, succinic, benzoic, salicylic,methanesulfonic, toluenesulfonic, trifluoroacetic, or pamoic acid, aswell as polymeric acids such as tannic acid or carboxymethyl cellulose,and salts with inorganic acids, such as hydrohalic acids (e.g.,hydrochloric acid), sulfuric acid, or phosphoric acid and the like. Anyprocedure for obtaining a pharmaceutically acceptable salt known to askilled artisan can be used.

In the practice of the method of the present invention, an effectiveamount of any one of the peptides of this invention or a combination ofany of the peptides of this invention or a pharmaceutically acceptablesalt thereof, is administered via any of the usual and acceptablemethods known in the art, either singly or in combination.Administration can be, for example, once a day, once every three days oronce a week. The compounds or compositions can thus be administeredorally (e.g., buccal cavity), sublingually, parenterally (e.g.,intramuscularly, intravenously, or subcutaneously), rectally (e.g., bysuppositories or washings), transdermally (e.g., skin electroporation)or by inhalation (e.g., by aerosol), and in the form or solid, liquid orgaseous dosages, including tablets and suspensions. The administrationcan be conducted in a single unit dosage form with continuous therapy orin a single dose therapy ad libitum. The therapeutic composition canalso be in the form of an oil emulsion or dispersion in conjunction witha lipophilic salt such as pamoic acid, or in the form of a biodegradablesustained-release composition for subcutaneous or intramuscularadministration.

Thus, the method of the present invention is practiced when relief ofsymptoms is specifically required or perhaps imminent. Alternatively,the method of the present invention is effectively practiced ascontinuous or prophylactic treatment.

Useful pharmaceutical carriers for the preparation of the compositionshereof, can be solids, liquids or gases; thus, the compositions can takethe form of tablets, pills, capsules, suppositories, powders,enterically coated or other protected formulations (e.g. binding onion-exchange resins or packaging in lipid-protein vesicles), sustainedrelease formulations, solutions, suspensions, elixirs, aerosols, and thelike. The carrier can be selected from the various oils including thoseof petroleum, animal, vegetable or synthetic origin, e.g., peanut oil,soybean oil, mineral oil, sesame oil, and the like. Water, saline,aqueous dextrose, and glycols are preferred liquid carriers,particularly (when isotonic with the blood) for injectable solutions.For example, formulations for intravenous administration comprisesterile aqueous solutions of the active ingredient(s) which are preparedby dissolving solid active ingredient(s) in water to produce an aqueoussolution, and rendering the solution sterile. Suitable pharmaceuticalexcipients include starch, cellulose, talc, glucose, lactose, talc,gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodiumstearate, glycerol monostearate, sodium chloride, dried skim milk,glycerol, propylene glycol, water, ethanol, and the like. Thecompositions may be subjected to conventional pharmaceutical additivessuch as preservatives, stabilizing agents, wetting or emulsifyingagents, salts for adjusting osmotic pressure, buffers and the like.Suitable pharmaceutical carriers and their formulation are described inRemington's Pharmaceutical Sciences by E. W. Martin. Such compositionswill, in any event, contain an effective amount of the active compoundtogether with a suitable carrier so as to prepare the proper dosage formfor proper administration to the recipient.

The dose of a compound of the present invention depends on a number offactors, such as, for example, the manner of administration, the age andthe body weight of the subject, and the condition of the subject to betreated, and ultimately will be decided by the attending physician orveterinarian. Such an amount of the active compound as determined by theattending physician or veterinarian is referred to herein, and in theclaims, as an “effective amount”. For example, the dose for intranasaladministration is typically in the range of about 0.001 to about 0.1mg/kg body weight. In humans, the preferred subcutaneous dose based onpeptide content is from about 0.001 mg to about 100 mg; preferably fromabout 0.1 mg to about 15 mg. For the active pharmaceutical ingredient(API), it would range from about 0.015 mg to about 100 mg; preferablyfrom about 1 mg to about 100 mg.

The invention will now be further described in the Examples whichfollow, which are intended as an illustration only and do not limit thescope of the invention.

EXAMPLES

A 1 L round-bottom flask equipped with a magnetic stirrer, dean-starktrap, reflux condenser and argon (or nitrogen) inlet bubbler was chargedwith 100 g (3.34 mmol) of mPEG 30 kDa (obtained from Nippon Oil and Fat)and 500 mL of toluene. The PEG solution in toluene was azeotropicallydried by distilling off 250 mL of toluene then the solution was cooledto room temperature. To the solution 200 mL of anhydrous dichloromethanewas added, the solution was cooled to 0-5° C. and 0.67 mL (4.84 mmol) oftriethylamine and 0.33 mL (4.34 mmol) of methanesulfonyl chloride wereadded dropwise using a syringe through a rubber septa. The mixture wasstirred for 2 hrs at ca. 4° C. and then stirred at room temperatureovernight under argon gas.

The mixture was concentrated on a rotary evaporator and was filteredthrough a coarse glass frit to remove salts. (Caution: warm up the glassfrit during the filtration to prevent the solution from solidifying).The product was precipitated by the addition of ca. 1800 ml of coldisopropyl alcohol and diethyl ether (30:70, v/v). The product wascollected and dried under vacuum at room temperature overnight to give90 g (90%) of a white solid.

A 2-L, round-bottom flask equipped with a magnetic stirrer and argoninlet bubbler was charged with 90 g (3 mmol) of mPEG 30 kDa mesylate 2prepared above and 1600 mL of ammonium hydroxide aqueous solution (30%,v/v). To this solution 160 g of ammonium chloride was added. Thesolution was warmed carefully to dissolve all of the PEG mesylate. Theresulting solution was stirred at room temperature for 48 h whileventing excess gases through a bubbler to prevent pressure buildup inthe reaction flask.

After the reaction was complete, 160 g (10 wt %) of sodium chloride wasadded and the mixture was extracted with 3×200 mL=1200 mL ofdichloromethane. The combined organic extracts were dried over anhydroussodium sulfate for about 1 h, filtered and concentrated on a rotaryevaporator. The product was precipitated by addition of 1800 mL of colddiethyl ether, filtered and dried under vacuum at room temperatureovernight to give 85 g (94%) of 3 as a white solid.

A 1 L, round-bottom flask equipped with a magnetic stirrer and argoninlet bubbler was charged with 60 g (2.00 mmol) of mPEG 30 kDa amine 3and 500 mL of anhydrous acetonitrile. The solution was cooled down toca. 4° C., then 2 g (20.00 mmol) of succinic anhydride in 50 mL ofanhydrous acetonitrile was added slowly using addition funnel. Thereaction mixture was stirred at room temperature overnight under anargon gas flow.

After the reaction was finished, the solvent was evaporated to drynessby rotary evaporator. Then, the product was dissolved in 400 mL ofwater. The pH of the solution was adjusted to 7.0 with 1 M NaOH solutionand stirred for 1 h while maintaining pH at 7.0. To this solution 40 g(10 wt. %) of sodium chloride was added and the pH was adjusted to ˜4.2with 6 N HCl solution. The resulting aqueous mixture was extracted with200, 100, 50 mL=350 mL of dichloromethane. The combined organic extractswere dried over anhydrous sodium sulfate for about 1 h. The sodiumsulfate was filtered off the filtrate was concentrated on a rotaryevaporator. Precipitate the product in 1 L of cold diethyl ether.Collect the product and dry it under vacuum at room temperatureovernight to give 56 g (93%) of 4 as a white solid.

A 500-mL, round-bottom flask equipped with a magnetic stirrer and argoninlet bubbler was charged with 56 g (1.87 mmol) of mPEG 30 kDaSuccinamide Acid 4 and 500 mL of anhydrous dichloromethane. To thissolution 0.24 g (2.05 mmol) of N-hydroxysuccinimide and 0.46 g (2.24mmol) of 1,3-dicyclohexylcarbodiimide were added slowly. The reactionmixture was stirred at room temperature overnight under argon gas flow.

After the reaction was complete, the mixture was evaporated to drynesson a rotary evaporator. Then, the product was dissolved in 200 mL ofanhydrous toluene and the solution was filtered through a pre-warmedcoarse glass frit laid with a pad of celite. The product wasprecipitated by addition of 1200 mL of cold anhydrous isopropyl alcoholand diethyl ether (30:70, v/v). The product was collected and driedunder vacuum at room temperature overnight to give 20 g (80%) of 5 as awhite solid.

PREPARATION OF PREFERRED COMPOUNDS Example 1 Preparation ofFmoc-Linker-BHA Resin

Benzhydrylamine copolystyrene-1% divinylbenzene cross-linked resin (10.0g, 9.3 mequiv, 100-200 ASTM mesh, Advanced ChemTech) was swelled in 100mL CH₂Cl₂, filtered and washed successively with 100 ml each of CH₂Cl₂,6% DIPEA/CH₂Cl₂ (two times), CH₂Cl₂ (two times). The resin was treatedwithp-((R,S)-α-(1-(9H-fluoren-9-yl)-methoxyformamido)-2,4-dimethoxybenzyl)-phenoxyaceticacid (Fmoc-Linker) (7.01 g, 13.0 mmole), N-hydroxybenzotriazole (2.16 g,16.0 mmole), and diisopropyl-carbodiimide (2.04 ml, 13.0 mmol) in 100 mL25% DMF/CH₂Cl₂ for 24 hours at room temperature. The resin was filteredand washed successively with 100 ml each of CH₂Cl₂ (two times),isopropanol (two times), DMF, and CH₂Cl₂ (three times). A KaiserNinhydrin analysis was negative. The resin was dried under vacuum toyield 16.12 g of Fmoc-Linker-BHA resin. A portion of this resin (3.5 mg)was subjected to Fmoc deprotection and quantitative UV analysis whichindicated a loading of 0.56 mmol/g.

Example 2 Protocol for the Synthesis of Peptides by Applied Biosystem433A synthesizer Using Fluorenylmethyloxycarbonyl (Fmoc) Chemistry

For a 0.25 mmol scale peptide synthesis by Applied Biosystem 433Asynthesizer (Foster City, Calif.), the FastMoc 0.25 mmole cycles wereused with either the resin sampling or non resin sampling, 41 mLreaction vessel. The Fmoc-amino acid resin was dissolved with 2.1 g NMP,2 g of 0.45M HOBT/HBTU in DMF and 2M DIEA, then transferred to thereaction vessel. The basic FastMoc coupling cycle was represented by“BADEIFD,” wherein each letter represents a module (as defined byApplied Biosystems). For example:

B represents the module for Fmoc deprotection using 20% Piperidine/NMPand related washes and readings for 30 min (either UV monitoring orconductivity); A represents the module for activation of amino acid incartridges with 0.45 M HBTU/HOBt and 2.0 M DIEA and mixing with N₂bubbling; D represents the module for NMP washing of resin in thereaction vessel; E represents the module for transfer of the activatedamino acid to the reaction vessel for coupling; I represents the modulefor a 10 minute waiting period with vortexing on and off of the reactionvessel; and F represents the module for cleaning cartridge, coupling forapproximately 10 minutes and draining the reaction vessel. Couplingswere typically extended by addition of module “I” once or multipletimes. For example, double couplings were run by performing theprocedure “BADEIIADEIFD.” Other modules were available such as c formethylene chloride washes and “C” for capping with acetic anhydride.Individual modules were also modifiable by, for example, changing thetiming of various functions, such as transfer time, in order to alterthe amount of solvent or reagents transferred. The cycles above weretypically used for coupling one amino acid. For synthesizing tetrapeptides, however, the cycles were repeated and strung together. Forexample, BADEIIADEIFD was used to couple the first amino acid, followedby BADEIIADEIFD to couple the second amino acid, followed byBADEIIADEIFD to couple the third amino acid, followed by BADEIIADEIFD tocouple the fourth amino acid, followed by BIDDcc for final deprotectionand washing.

Example 3 Preparation ofH-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-The-Arg-Gln-Arg-Tyr-NH₂(PY₃₋₃₆)

The above peptide was synthesized using Fmoc chemistry on an AppliedBiosystem 433A synthesizer. The synthesizer was programmed for doublecoupling using the modules described in Example 2. The synthesis wascarried out on a 0.25 mmol scale using the Fmoc-Linker-BHA resin (450mg, 0.25 mmol) from Example 1. At the end of the synthesis, the resinwas transferred to a reaction vessel on a shaker for cleavage. Thepeptide was cleaved from the resin using 13.5 mL 97% TFA/3% H₂O and 1.5mL triisopropylsilane for 180 minutes at room temperature. Thedeprotection solution was added to 100 mL cold ET₂O, and washed with 1mL TFA and 30 mL cold ET₂O to precipitate the peptide. The peptide wascentrifuged 2×50 mL polypropylene tubes. The precipitates from theindividual tubes were combined in a single tube and washed 3 times withcold ET₂O and dried in a desiccator under house vacuum.

The crude material was purified by preparative HPLC on a PursuitC18-Column (250×50mm, 10 μm particle size) and eluted with a lineargradient of 2-70% B (buffer A: 0.1% TFA/H₂ 0; buffer B: 0.1% TFA/CH3CN)in 90 min., flow rate 60 mL/min,and detection 220/280 nm. The fractionswere collected and were checked by analytical HPLC. Fractions containingpure product were combined and lyophilized to yield 151 mg (15%) of awhite amorphous powder. (ES)+-LCMS m/e calculated (“calcd”) forC₁₈₀H₂₇₉N₅₃O₅₄ 4049.55. found 4050.40.

Example 4 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following the procedure inExample 3 to yield 48 mg (9%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C₉₈H₁₅₅N₃₃0₂₁ 2131.53. found 2130.56.

Example 5 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis (N-methyl Arg was inserted in position 35 ofthe sequence) and purification by following the procedure in Example 3to yield 32 mg (6%) of white amorphous powder. (ES)+-LCMS m/e calcd forC₉₉H₁₅₅N₃₃O₂₁ 2143.56. found 2143.50.

Example 6 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-m-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 38.5 mg (7%) of white amorphous powder. (ES)+-LCMSm/e calcd for C99H155N33O21 2143.5477. found 2143.50.

Example 7 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-3-iodo-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 41 mg (7%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C99H154IN33O21 2269.44. found 2269.20.

Example 8 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-3,5 diF-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 28mg (5%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C99H153F2N33O21 2179.52. found 2179.46.

Example 9 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-2,6 diF-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 49.3 mg (9%) of white amorphous powder. (ES)+-LCMSm/e calcd for C99H153F2N33O21 2179.53. found 2179.50.

Example 10 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-2,6 diMe-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 13.5 mg (3%) of white amorphous powder. (ES)+-LCMSm/e calcd for C101H159N33O21 2171.60. found 2171.40.

Example 11 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-4Methoxy-Phe-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 72 mg (13%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C100H157N33O21 2157.57. found 2157.58.

Example 12 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-Phe-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 85.3 mg (16%) of white amorphous powder. (ES)+-LCMSm/e calcd for C99H155N33O20 2127.55. found 2127.53.

Example 13 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-4amino-Phe-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 51.4 mg (10%) of white amorphous powder. (ES)+-LCMSm/e calcd for C99H156N34O20 2142.56. found 2142.55.

Example 14 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-4F-Phe-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 35 mg (7%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C99H154FN33O20 2145.54. found 2145.51.

Example 15 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-4-(CH2OH)-Phe-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 24 mg (4%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C100H1571N33O21 2157.57. found 2157.56.

Example 16 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-4-trifluoromethyl-Phe-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 81 mg (15%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C100H154F3N33O20 2195.54. found 2195.51.

Example 17 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-3Fluoro-Phe-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 84 mg (16%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C99H154FN33O20 2145.54. found 2145.53.

Example 18 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-2,3,4,5,6Pentafluoro-Phe-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 89 mg (16%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C99H150FN33O20 2217.50. found 2217.48.

Example 19 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-3,4-dichloro-Phe-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 46 mg (8%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C99H153C12N33O20 2196.44. found 2196.41.

Example 20 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-Cha-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 49 mg (9%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C106H162N34O21 2248.69. found 2248.71.

Example 21 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-Trp-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 57 mg (10%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C108H157N35O21 2281.68. found 2281.67.

Example 22 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-1-Nal-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 45 mg (8%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C103H157N33O20 2177.61. found 2177.59.

Example 23 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe)Arg-2-Nal-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 43 mg (8%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C103H157N33O20 2177.60. found 2177.58.

Example 24 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-C-α-methylTyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 35.1 mg (7%) of white amorphous powder. (ES)+-LCMSm/e calcd for C99H155N33O21 2143.55. found 2143.56.

Example 25 Preparation ofH-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 130 mg (23%) of white amorphous powder. (ES)+-LCMSm/e calcd for C104H154N34O21 2216.60. found 2216.62.

Example 26 Preparation ofH-Ile-Nle-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 84 mg (15%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C104H153N33O21 2201.59. found 2201.56.

Example 27 Preparation ofAc-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-2,6-F2-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 24 mg (4%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C106H154F2N34O22 2099.49. found 2100.3.

Example 28 Preparation ofAc-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 68 mg (12%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C106H156N34O22 2258.64. found 2258.61.

Example 29 Preparation ofPentoyl-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 67 mg (12%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C109H162N34O22 2300.72. found 2300.69.

Example 30 Preparation ofTrimethylacetyl-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 6 mg (1%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C109H162F2N34O22 2300.72. found 2300.68.

Example 31 Preparation ofCyclohexylacetyl-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 15 mg (3%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C112H166N34O22 2340.79. found 2340.81.

Example 32 Preparation ofBenzoyl-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 23 mg (4%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C111H158N34O22 2320.71. found 2320.68.

Example 33 Preparation ofAdamantoyl-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

Fmoc- Linker-BHA resin (450 mg, 0.25 mmol) from Example 1 was subjectedto solid phase synthesis and purification by following procedure inExample 3 to yield 29 mg (5%) of white amorphous powder. (ES)+-LCMS m/ecalcd for C116H170N34O22 2392.86. found 2392.89.

Analytical Method for Examples 34-39 and 41

The test and control articles were analyzed using the followingreversed-phase HPLC/UV procedure:

-   Autosampler Alliance Waters 2690 Separation Module-   Injection Volume 10 μL-   Injector Temperature Ambient-   Detector Waters 996 Photodiode Array Detector-   Detector Wavelength 280 nm-   Column Agilent Eclipse XDB-C8, 5 micron, 150 mm×4.6 mm i.d.    PN:99367-906-   Color Temperature 25° C.-   Flow Rate 1.0 mL/minute (˜1000 psi)-   Mobile Phase A Water containing 0.05% TFA-   Mobile Phase B Acetonitrile containing 0.05% TFA-   Run Time Approximately 30 minutes-   Sample Preparation Approximately 0.2-0.5 mg/ml-   Diluent Deionized water

Mobile Phase Gradient Condition 1 (RP-HPLC1): Time, minutes % MobilePhase A % Mobile Phase B Condition 0 95 5 Linear ramp 20 5 95 26 95 5Equilibrium

Mobile Phase Gradient Condition 2 (RP-HPLC2): Time, minutes % MobilePhase A % Mobile Phase B Condition 0 85 15 Linear ramp 20 25 75 26 85 15Equilibrium

Example 34 Preparation of mixture of((PEG-30,000)CH₂CH₂CO)Ile-Lys-Pqa-Arg-His-Tyr-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂andIle((PEG-30,000)CH₂CH₂CO)(ε)Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

where n = ˜675 and

where n = ˜675 Analytical Method Result RP-HPLC1 - rxn mixture 69.8%conversion RP-HPLC1 - purified 10.1 min Retention time MALDI-TOF MSAverage Mass = 33.9 kDa

Twenty-five mg of peptide from Example 25 was weighed out and dissolvedin 50 mM Borate, pH 7.5 buffer. 500 mg 30 kDa PEG-succinimidylproprionic acid (purchased from Nektar) was weighed to achieve a 2:1PEG: peptide molar ratio and added to the dissolved peptide. Thereaction mixture was agitated at room temperature overnight before itwas diluted 10-fold in 20 mM NaOAc, pH 4.5 buffer and purified by cationexchange chromatography on SP-Sepharose FF. FIG. 1 is an HPLCchromatogram of the reaction mixture. The reaction yielded 69.8% of 30kDa peptide.

Mono-pegylated PYY peptide was eluted using a step NaCl gradient.Typically, the desired mono-pegylated peptide eluted with 250 mM NaCl.The eluted PEG-PYY-like peptide was concentrated in an Amiconultrafiltration cell using a 10 kDa MW cutoff membrane. It was thendiafiltered 10-fold once with PBS.

Concentrated peptide of Example 34 was submitted for analysis, assayedand stored at −20 C. FIG. 2 is an HPLC chromatogram of purified 30 kDaPEG-PYY peptide (RT=0.1 min). Purity of 30 kDa peptide was determined tobe >97%. And FIG. 3 is a graph representing a MALDI-TOF of 30 kDaPEG-PYY peptide, which was performed to confirm the molecular weight.

A combination of methods was used to determine the PEG modificationsites. These included, MALDI TOF MS, reversed phase HPLC, proteolyticdigestion and N-terminal sequencing (Edman). The results from theseanalyses showed that the majority of the PEG is attached through theε-amino group of the lysine in position (R2) of the peptide.

Example 35 Preparation of a mixture of((PEG-40,000)CO)Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂andIle((PEG-40,000)CO)(ε)Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

where n = ˜900 and

where n = ˜900 Analytical Method Result RP-HPLC1 - rxn mixture 60.4%conversion RP-HPLC1 - purified 10.1 min Retention time MALDI-TOF MSAverage Mass = 41.9 kDa

Twenty-five mg of peptide from Example 25 was weighed out and dissolvedin 50 mM Borate, pH 8.0 buffer. 319 mg 40 kDa PEG-benzotriazolecarbonate was weighed to achieve a 0.8:1 PEG: peptide molar ratio andadded to the dissolved peptide. The reaction mixture was agitated atroom temperature for 1 h before it was diluted 10-fold in 20 mM NaOAc,pH 4.5 buffer and purified by cation exchange chromatography onSP-Sepharose FF. FIG. 4 is an HPLC chromatogram of the reaction mixture.The reaction yielded 60.4% of 40 kDa peptide.

Mono-pegylated PYY peptide was eluted using a step NaCl gradient.Typically, the desired mono-pegylated peptide eluted with 250 mM NaCl.The eluted PEG-PYY-like peptide was concentrated in an Amiconultrafiltration cell using a 10 kDa MW cutoff membrane. It was thendiafiltered 10-fold once with PBS.

Concentrated peptide of Example 35 was submitted for analysis, assayedand stored at −20 C. FIG. 5 is an HPLC chromatogram of purified 40 kDaPEG-PYY peptide (RT=10.1 min). Purity of 40 kDa peptide was determinedto be >90%. And FIG. 6 is a graph representing a MALDI-TOF of 40 kDaPEG-PYY peptide, which was performed to confirm the molecular weight.

Example 36 Preparation of((PEG-30,000)CH₂CH₂NHCOCH₂CH₂CO)Ile-Nle-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

where n = ˜675 Analytical Method Result RP-HPLC1 - rxn mixture 94.1%conversion RP-HPLC1 - purified 110.4 min Retention time MALDI-TOF MSAverage Mass = 34.2 kDa

Thirteen mg of peptide from Example 26 was weighed out and dissolved in50 mM Borate, pH 8.0 buffer. 624 mg 30 kDa PEG-succinimidyl succinamidewas weighed to achieve a 4:1 PEG: peptide molar ratio and added to thedissolved peptide. The reaction mixture was agitated at room temperaturefor 2 h before it was diluted 10-fold in 20 mM NaOAc, pH 4.5 buffer andpurified by cation exchange chromatography on SP-Sepharose FF(Amersham). FIG. 7 is an HPLC chromatogram of the reaction mixture. Thereaction yielded 94.1% of 30 kDa peptide.

Mono-pegylated PYY peptide was eluted using a step NaCl gradient.Typically, the desired mono-pegylated PYY peptide eluted with 250 mMNaCl. The eluted PEG-PYY-like peptide was concentrated in an Amiconultrafiltration cell using a 10 kDa MW cutoff membrane. It was thendiafiltered 10-fold once with PBS. Concentrated peptide was submittedfor analysis, assayed and stored at −20 C. FIG. 8 is an HPLCchromatogram of purified 30 kDa PEG-PYY peptide (10.4 min). Purity of 30kDa peptide was determined to be >90%. And FIG. 9 represents a MALDI-TOFof 30 kDa PEG-PYY peptide that was performed to confirm the molecularweight.

Example 37 Preparation of((PEG-30,000)CH(CH₃)CH₂CO)Ile-Nle-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

where n = ˜675 Analytical Method Result RP-HPLC1 - rxn mixture 93.4%conversion RP-HPLC1 - purified 110.5 min Retention time MALDI-TOF MSAverage Mass = 34.6 kDa

1.25 mg of peptide from Example 26 was weighed out and dissolved in 50mM Borate, pH 8.0 buffer. 62 mg 30 kDa PEG-succinimidyl beta-SBA wasweighed to achieve a 4:1 PEG: peptide molar ratio and added to thedissolved peptide. The reaction mixture was agitated at room temperaturefor 2 h before it was diluted 10-fold in 20 mM NaOAc, pH 4.5 buffer andpurified by cation exchange chromatography on SP-Sepharose FF(Amersham). FIG. 10 is an HPLC chromatogram of the reaction mixture. Thereaction yielded 93.4% of 30 kDa peptide.

Mono-pegylated PYY peptide was eluted using a step NaCl gradient.Typically, the desired mono-pegylated PYY peptide eluted with 250 mMNaCl. The eluted PEG-PYY-like peptide was concentrated in an Amiconultrafiltration cell using a 10 kDa MW cutoff membrane. It was thendiafiltered 10-fold once with PBS. Concentrated peptide was submittedfor analysis, assayed and stored at −20 C. FIG. 11 is an HPLCchromatogram of purified 30 kDa PEG-PYY peptide (10.5 min). Purity of 30kDa peptide was determined to be >90%. And FIG. 12 represents aMALDI-TOF of 30 kDa PEG-PYY peptide that was performed to confirm themolecular weight.

Example 38 Preparation ofAc-Ile(PEG-30,000)CH₂CH₂CO(ε)Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

where n = ˜675 Analytical Method Result RP-HPLC1 - rxn mixture 83.3%conversion RP-HPLC1 - purified 9.5 min Retention time MALDI-TOF MSAverage Mass = 34.6 kDa

One hundred mg of peptide from Example 28 was weighed out and dissolvedin 50 mM Borate, pH 8.0 buffer. 1.8 g 30 kDa PEG-succinimidyl proprionicacid (purchased from Nektar) was weighed to achieve a 1.5:1 PEG: peptidemolar ratio and added to the dissolved peptide. The reaction mixture wasagitated at room temperature overnight before it was diluted 10-fold in20 mM NaOAc, pH 4.5 buffer and purified by cation exchangechromatography on SP-Sepharose FF. FIG. 13 is an HPLC chromatogram ofthe reaction mixture. The reaction yielded 83.3% of 30 kDa peptide.

Mono-pegylated PYY peptide was eluted using a step NaCl gradient.Typically, the desired mono-pegylated peptide eluted with 250 mM NaCl.The eluted PEG-PYY-like peptide was concentrated in an Amiconultrafiltration cell using a 10 kDa MW cutoff membrane. It was thendiafiltered 10-fold once with PBS.

Concentrated peptide of Example 38 was submitted for analysis, assayedand stored at −20 C. FIG. 14 is an HPLC chromatogram of purified 30 kDaPEG-PYY peptide (RT=9.5 min). Purity of 30 kDa peptide was determined tobe >95%. And FIG. 15 is a graph representing a MALDI-TOF of 30 kDaPEG-PYY peptide, which was performed to confirm the molecular weight.

Example 39 Preparation ofAc-Ile((PEG-30,000)CH₂CH₂NHCOCH₂CH₂CO)(ε)Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

where n = ˜675 Analytical Method Result RP-HPLC1 - rxn mixture 81.4%conversion RP-HPLC1 - purified 9.5 min Retention time MALDI-TOF MSAverage Mass = 33.6 kDa

One hundred mg of peptide from Example 28 was weighed out and dissolvedin 50 mM Borate, pH 8.0 buffer. 3.6 g 30 kDa PEG-succinimidylsuccinamide was weighed to achieve a 3:1 PEG: peptide molar ratio andadded to the dissolved peptide. The reaction mixture was agitated atroom temperature overnight before it was diluted 10-fold in 20 mM NaOAc,pH 4.5 buffer and purified by cation exchange chromatography onSP-Sepharose FF. FIG. 16 is an HPLC chromatogram of the reactionmixture. The reaction yielded 81.4% of 30 kDa peptide.

Mono-pegylated PYY peptide was eluted using a step NaCl gradient.Typically, the desired mono-pegylated peptide eluted with 250 mM NaCl.The eluted PEG-PYY-like peptide was concentrated in an Amiconultrafiltration cell using a 10 kDa MW cutoff membrane. It was thendiafiltered 10-fold once with PBS.

Concentrated peptide of Example 39 was submitted for analysis, assayedand stored at −20 C. FIG. 17 is an HPLC chromatogram of purified 30 kDaPEG-PYY peptide (RT=9.5 min). Purity of 30 kDa peptide was determined tobe >97%. And FIG. 18 is a graph representing a MALDI-TOF of 30 kDaPEG-PYY peptide, which was performed to confirm the molecular weight.

Example 40 Preparation ofFmoc-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂

55.0 g Fmoc-linker BHA @0.45 mm/g purchased from AnaSpec Inc. cat #408/452-5055) was subjected to solid phase peptide synthesis andpurification by the following procedure:

Fmoc protected amino acids were couple via DIC/ROBt in 25% excess. (55.0g×0.45 mm/g×1.25 eqv=31.0 mm). All deprotection was 2×10 min with 20%piperdine in DMF at approximately 10 ml/g (adjusted as volume ofpeptide-resin increases). After deprotection the peptide resin waswashed 4× DMF (20 volumes).

All couplings were with ˜40 mL DIC (˜6 eqv) and 4.5 g HOBt (1.25 eqv).After coupling the resin solution was sampled ˜0.25 ml washed withCH₂Cl₂ and checked via ninhydrin for completion. After coupling toNMe-Arg and Pqa, the resin was washed with CH₂Cl₂ and checked with 3drops of 2% Chlorinal in DMAc and 3 drops 2% Acetaldehyde in DMAc (withno change in the yellow solution indicative of no secondary amine andblue to black beads indicative of incomplete coupling). If couplingswere judged to be incomplete DIEA was added to the coupling solution andcontinued for 1 hr further. When complete, the resin was washed 4×DMF(20 volumes).

Fmoc-Tyr(But)-OH: 14.0 g. Fmoc-NMeArg(Mtr)-OH: 20.0 g. Fmoc-Gln(Trt)-OH:18.5 g .Fmoc-Arg(Pbf)-OH: 20.0 g. Fmoc-Thr(But)-OH: 18.0 g. Fmoc-Val-OH:10.5 g. Fmoc-Trp-OH: 13.0. Fmoc-Asn(Trt)-OH: 18.0 g. Fmoc-Leu-OH 11.0 g,Fmoc-Tyr(But)-OH 14.0 g, Fmoc-His(Trt)-OH 20.0 g, Fmoc-Arg(Pbf)-OH 20.0g, Fmoc-Pqa-OH 15.4 g, Fmoc-Lys(Boc)-OH 14.1 g, Fmoc-Ile-OH 11.0 g.

The peptide resin was washed 3×DMF, 3×CH₂Cl₂ and 3×MeOH and dried undersuction to obtain 115.38 g peptide resin. The resin was split into6×19.25 g batches for TFA cleavage.

19.25 g of peptide resin was cleaved with 8.0 mL 1:1:1 DTE; Anisole;Thioanisloe, 8.0 mL iPr₃SiH, 8.0 mL H₂O and 200 mL TFA/for 6 hours (6:30AM to 13:30 PM), followed by precipitation in 2.0 L cold (−20° C.) Et2O.The precipitate was collected by centrifugation in 8×50 mL polypropylenecentrifuge tubes and washed 3' cold Et₂O. The precipitate was then driedunder house vacuum overnight to obtain 6.5 g of crude peptide. The totalamount of crude peptide after 6 deprotection was 38.71 g.

Purification of crude peptide was performed on Shimadzu LC-8A system byhigh performance liquid chromatography (HPLC) on a reverse phase PursuitC-18 Column (50×250 mm. 300 A°, 10 um). 38.71 g of crude was purified in36 preps. Each time approximately 1.1 g of crude peptide was dissolvedin a minimum amount of water and acetonitrile and was injected in acolumn. Gradient elution was generally started at 20% B buffer, 20%-70%B over 70 minutes, (buffer A: 0.1% TFA/H2O, buffer B: 0.1% TFA/CH3CN) ata flow rate of 50 ml/min. UV detection was made at 220/280 nm. Thefractions containing the products were separated and purity was judgedon Shimadzu LC-10AT analytical system using reverse phase Pursuit C18column (4.6×50mm) at a flow rate of 2.5 ml/min., gradient (20-70%) over10 min.[buffer A: 0.1% TFA/H2O, buffer B: 0.1% TFA/CH3CN)]. Fractionsjudged to be of high purity were pooled and lyophilized to yield whiteamorphous powder. Lyophilized product from thirty-six preps was combinedand lyophilized again to yield 8.233 g of pure peptide (12.6%). Purityof the final product was checked again by analytical HPLC on a reversedphase column as stated above and was approximately 95-99%. (ES)+-LCMSm/e calcd for C119H164N34O23 2438.85, found 2438.84.

Example 41 Preparation ofIle((PEG-30,000)CH₂CH₂NHCOCH₂CH₂CO)(ε)Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)-Tyr-NH₂

n = ˜675 Analytical Method Result RP-HPLC2 93.8% conversion - protectedPEG-peptide 89.5% yield overall - deprotected PEG- peptide RP-HPLC2 -purified 10.1 min Retention time MALDI-TOF MS Average Mass = 35 kDa

1.8 g of peptide from Example 40 was weighed out and dissolved in 50 mMBorate, pH 7.5 buffer. 37.5 g 30 kDa PEG-succinimidyl succinamide wasweighed to achieve an approximate 2:1 PEG: peptide molar ratio and addedto the dissolved peptide. The reaction mixture was stirred at roomtemperature for 2 h. The pegylated peptide was deprotected by addingpiperidine (20%) to the reaction mixture for 1 h at room temperature.The reaction mixture was placed on ice and acidified by slow addition ofglacial acetic acid (20%). The reaction mixture was then diluted 10-foldin 20 mM NaOAc, pH 4.5 buffer and purified by cation exchangechromatography on SP-Sepharose FF. FIG. 19 is an HPLC chromatogram ofthe reaction mixture. The reaction yielded 93.8% of protected pegylatedpeptide. FIG. 20 is an HPLC chromatogram of the deprotected reactionmixture, whereby the overall yield of pegylated deprotected peptide was89.5%.

Mono-pegylated PYY peptide was eluted using a step NaCl gradient.Typically, the desired mono-pegylated peptide eluted with 175 mM NaCl.The eluted PEG-PYY-like peptide was concentrated in an Amiconultrafiltration cell using a 10 kDa MW cutoff membrane. It was thendiafiltered 10-fold once with PBS.

Concentrated peptide was submitted for analysis, assayed and stored at−20 C. FIG. 21 is an HPLC chromatogram of purified 30 kDa PEG-PYYpeptide (RT=10.1 min). Purity of 30 kDa peptide was determined tobe >95%. And FIG. 22 is a graph representing a MALDI-TOF of 30 kDaPEG-PYY peptide, which was performed to confirm the molecular weight.

Example 42 Calcium Flux Assay

HEK-293 cells stably transfected with the G protein chimera Gaqi9 andthe hygromycin-B resistance gene were further transfected with the humanNPY2 receptor and G418 antibiotic selection. Following selection in bothhygromycin-B and G418, individual clones were assayed for their responseto PYY. The transfected cells (HEK293/hNPY2R) were cultured in DMEMmedium supplemented with 10% fetal bovine serum, 50 μg/ml hygromycin-B,2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 250μg/ml G418. Cells were harvested with trypsin-EDTA and counted usingViaCount reagent. The cell suspension volume was adjusted to 4.8×10⁵cells /ml with complete growth media. Aliquots of 25 μl were dispensedinto 384-well Poly-D Lysine coated black/clear microplates (Falcon) andthe microplates were placed in a 37° C. CO₂ incubator overnight.

Loading Buffer (Calcium-3 Assay Kit, Molecular Devices) was prepared bydissolving the contents of one vial (Express Kit) into 1000 ml Hank'sBalanced Salt Solution (HBSS) containing 20 mM HEPES and 5 mMprobenecid. Aliquots (25 μl) of diluted dye were dispensed into the cellplates and the plates were then incubated for 1 h at 37° C.

During the incubation, test compounds were prepared at 3.5× the desiredconcentration in HBSS (20 mM HEPES)/0.05% BSA/1% DMSO and transferred toa 384-well plate for use on FLIPR® (FLIPR, Fluorescent Imaging PlateReader, is a registered trademark of Molecular Devices Corp.).

After incubation, both the cell and compound plates were brought to theFLIPR® and 20 μL of the diluted compounds were transferred to the cellplates by the FLIPR®. During the assay, fluorescence readings were takensimultaneously from all 384 wells of the cell plate every 1.5 seconds.Five readings were taken to establish a stable baseline, and then 20 μlof sample was rapidly (30 μl/sec) and simultaneously added to each wellof the cell plate. The fluorescence was continuously monitored before,during and after sample addition for a total elapsed time of 100seconds. Responses (increase in peak fluorescence) in each wellfollowing addition was determined. The initial fluorescence reading fromeach well, prior to ligand stimulation, was used as a zero baselinevalue for the data from that well. The responses were expressed as apercentage of maximal response of the positive control.

Example 43 Cyclic AMP Assay

In this example, the following materials were used: 384-well plate;Tropix cAMP-Screen Kit: (Applied Biosystems, Cat. #T1504); forskolin(Calbiochem Cat. #344270); cells: HEK293/hNPY2R cells; plating medium:DMEM/F12 w/o phenol red (Gibco Cat #1133032); 10% heat inactivated FBS(Gibco Cat. #10082-147); 1% Penicillin/Streptomycin (Gibco Cat.#15140-122); 500 mg/ml G418 (Geneticin, Gibco Cat. #11811-031).

HEK293/hNPY2R cells were plated at a density of 10⁴ cells/well in a384-well plate using Multi-drop dispenser and the plates were incubatedovernight at 37° C. The next day, cells that reached 75-85% confluencewere used in the experiment. The media and reagents were warmed to roomtemperature. Before the dilutions were prepared, the stock solution ofY2-receptor ligands and controls in dimethyl sulphoxide (DMSO, SigmaCat# D2650) was allowed to warm up to 32° C. for 5-10 min. The dilutionswere performed using incubation media [DMEM/F12 media containing 0.5 mM3-isobutyl-1-methylxanthine (IBMX, Calbiochem Cat #410957) and 0.5 mg/mlBSA (Sigma Cat #A8806)]. The final concentrations of DMSO and forskolinin the incubation medium were 1.1% and 5 μM, respectively.

The plating media was removed by gentle inversion of the 384-well plateon a paper towel and replaced with incubation medium (50 μ/well)containing various concentrations of Y2-receptor ligands (fourreplicates/concentration). The plates were incubated at room temperaturefor 30 min. Following the 30 min treatment period, the incubation mediawas discarded and replaced with 50 μl/well of Assay Lysis Buffer(provided in the Tropix kit). The cells were lysed by incubating platesfor 45 min @ 37° C. The lysate (20 μl) was transferred into thepre-coated antibody plates (384-well) supplied in the Tropix kit. APconjugate (10 μl) and of anti-cAMP antibody (20 μl) was added to eachwell and the plates incubated on a shaker at room temperature for 1 h.The plates were washed 5 times with Wash Buffer (70 μl/well/wash) andthe plates tapped dry. CSPD/Saphire-II RTU substrate/enhancer solution(30 μl/well) was added and incubated for 45 min @ room temperature. Thesignal in each well was measured (1 sec/well) using a Luminometer(VICTOR-V).

The compounds of the present invention exhibited selectiveNeuropeptide-2 receptor activity in vitro, as demonstrated in thecalcium flux assay (FLIPR®; Example 42) and cyclic AMP assay (Example43). Summary of the in vitro results, EC₅₀ for Examples 3 to 39 and 41,are illustrated in Table 1 below: Y2R Y2R Y1R Y4R Y5R EC50 EC50 EC50EC50 EC50 (nM) (nM) (nM) (nM) (nM) Example Sequence FLIPR cAMP FLIPRFLIPR FLIPR 3 IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (3-36) 0.12 0.033 633238 265 4 IK-Pqa-RHYLNLVTRQRY 0.28 0.047 57 >5000 1887 5IK-Pqa-RHYLNLVTRQ(N-methyl)RY 2.3 0.42 >5000 >5000 >5000 6IK-Pqa-RHYLNLVTRQ(N-methyl)R (m-)Y 3.32 1.5 >5000 >5000 >5000 7IK-Pqa-RHYLNLVTRQ(N-methyl)R (3-I)Y 1.15 0.31 173 >5000 >5000 8IK-Pqa-RHYLNLVTRQ(N-methyl)R (3-5 di F)Y 0.15 0.36 >5000 >5000 >5000 9IK-Pqa-RHYLNLVTRQ(N-methyl)R (2-6 di F)Y 0.11 0.19 303 >5000 >5000 10IK-Pqa-RHYLNLVTRQ(N-methyl)R (2-6 di Me)Y 0.28 0.67 762 >5000 >5000 11IK-Pqa-RHYLNLVTRQ(N-methyl)RF(4-O-CH3) 0.74 0.55 189 >5000 >5000 12IK-Pqa-RHYLNLVTRQ(N-methyl)RF 1.54 0.69 422 >5000 >5000 13IK-Pqa-RHYLNLVTRQ(N-methyl)R(4-NH2)Phe 11.4 0.31 >5000 >5000 >5000 14IK-Pqa-RHYLNLVTRQ(N-methyl)R(4-F)Phe 0.45 0.96 >5000 >5000 2259 15IK-Pqa-RHYLNLVTRQ(N-methyl)R(4-CH2OH)Phe 0.46 0.45 >5000 >5000 >5000 16IK-Pqa-RHYLNLVTRQ(N-methyl)R(4-CF3)Phe 6.13 3.55 3268 >5000 729 17IK-Pqa-RHYLNLVTRQ(N-methyl)R(3-F)Phe 0.635 0.75 >5000 >5000 >5000 18IK-Pqa-RHYLNLVTRQ(N-methyl)R(2,3.4,5,6-Penta-F)Phe 11.92.5 >5000 >5000 >5000 19 IK-Pqa-RHYLNLVTRQ(N-methyl)R(3.4-diCl)Phe 4.031.47 >5000 >5000 >5000 20 IK-Pqa-RHYLNLVTRQ(N-methyl)RCha 0.4980.5 >5000 >5000 352 21 IK-Pqa-RHYLNLVTRQ(N-methyl)RW 0.4541.06 >5000 >5000 >5000 22 IK-Pqa-RHYLNLVTRQ(N-methyl)R(1)NaI 2.731.14 >5000 >5000 4772 23 IK-Pqa-RHYLNLVTRQ(N-methyl)R(2)NaI 4.112.4 >5000 >5000 2162 24 IK-Pqa-RHYLNLVTRQR-C-α-Me-Tyr 5.44 1.353259 >5000 25 IK-Pqa-RHYLNWVTRQ(N-methyl)RY 0.44 0.25 298 >5000 >5000 26INIe-Pqa-RHYLNWVTRQ(N-methyl)RY 8.1 0.108 >5000 >5000 >5000 27Ac-IK-Pqa-RHYLNWVTRQ(N-methyl)R (2-6 di F)Y 1.44 0.07 >5000 >5000 581228 Ac-IK-Pqa-RHYLNWVTRQ(N-methyl)RY 0.458 0.18 >5000 >5000 >5000 29Pentyl-IK-Pqa-RHYLNWVTRQ(N-methyl)RY 0.873 0.51 >5000 >5000 >5000 30Trimetyl acetyl-IK-Pqa-RHYLNWVTRQ(N-methyl)RY 1.1 0.26 >5000 >5000 >500031 Cyclohexyl-IK-Pqa-RHYLNWVTRQ(N-methyl)RY 1.67 1.37 >5000 >5000 >500032 Benzoyl-IK-Pqa-RHYLNWVTRQ(N-methyl)RY 0.79 0.66 >5000 >5000 >5000 33Adamtyl-IK-Pqa-RHYLNWVTRQ(N.methyl)RY 2.33 2.9 >5000 >5000 >5000 34 (PEG30,000 SPA)IK-Pqa-RHYLNWVTRQ(N-methyl)RY 37.9 18 >5000 >5000 >5000 35(PEG 40,000 BTC)-IK-Pqa-RHYLNWVTRQ(N-methylRY 590 14.7 >5000 >5000 >500036 (PEG 30,000)-SSA-INIe-Pqa-RHYLNWVTRQ(N-methyl)RY 2897.8 >5000 >5000 >5000 37 (PEG30,000)-beta-SBA-INIe-Pqa-RHYLNWVTRQ(N-methyl)RY 23922.4 >5000 >5000 >5000 38 Ac-Ile-Lys(PEG 30,000SPA)-Pqa-RHYLNWVTRQ(Ng-methyl)RY 549 24.4 >5000 >5000 >5000 39Ac-Ile-Lys(PEG 30,000 SSA)-Pqa-RHYLNWVTRQ(N-methyl)RV 107820.7 >5000 >5000 >5000 41 IK(PEG 30,000 SSA)-Pqa-RHYLNWVTRQ(N-methyl)RY13.5 9.8 >5000 >5000 >5000

Example 44 Chronic DIO Rat Studies

Male Sprague Dawley rats (7 weeks old) were obtained from Charles RiverLaboratories (USA) and housed in a temperature and humidity controlledenvironment with a 12 h light: 12 h dark cycle. The rats were given adlibitum access to a high fat chow diet (HFD; 60% of dietary kcal as fat,Research Diets D12492) and water throughout the study. Following 7 weekson the HFD, rats were sorted by body weight and caged singly. The ratswere dosed prior to onset of dark cycle with vehicle (s.c.) or compoundof Example 41 (1, 5 and 10 mg/kg, s.c.) once every two days for 3 weeks(N=6-8 rats/group). Body weight was recorded on days indicated in FIG.23.

Data Analysis:

All data shown are the mean±standard error (s.e.m.). Statisticalevaluation of the data was carried out using one-way ANOVA, followed byDunnett's test to determine where statistically significant differencesexisted between vehicle and drug treated groups. Differences wereconsidered statistically significant at P<0.05. Data analysis wascarried out with GraphPad software (GraphPad Prism).

Results:

Chronic administration of the compound of Example 41 (5 and 10 mg/kg,q48 hr, s.c.) in male DIO rats induced a significant decrease in bodyweight gain versus vehicle-treated animals following a 3-week treatmentperiod (FIG. 23).

Acute db/db Mouse Studies

Female db/db mice (C57BL/KsJ-Lep^(db/db), Jackson Laboratories, USA)were at 6 weeks of age when received. The mice were housed in atemperature and humidity controlled environment with a 12 h light: 12 hdark cycle, and given access to food (Purina rodent chow 5008) and waterad libitum. The mice (12-weeks old) were pre-bled 4 days prior to study,and those within a narrow range of fasted blood glucose levels wereselected for the study in order to minimize variability between vehiclecontrol and drug-treated groups. The mice were administered vehicle(s.c.) or the compound of Example 41 (0.3, 1 and 10 mg/kg, s.c.) 28 hrprior to the oral glucose tolerance test (N=10 mice/group). Bloodsamples were collected from tail clips following a 6 hr fast, fordetermination of baseline values (t=0 min). The mice were then gavagedwith an oral bolus of glucose (1 g/kg), and additional blood sampleswere collected at regular intervals (t=30, 60 and 120 min) for glucosemeasurement. To analyze the effects of the compound of Example 41 onoral glucose tolerance the absolute difference in blood glucose frombaseline (fasting blood glucose) was calculated for each time point. Thearea under the curve (AUC₀₋₁₂₀ min) was determined using the trapezoidmethod.

Data Analysis:

All data shown are the mean±standard deviation (s.d.). Statisticalevaluation of the data was carried out using one-way ANOVA, followed byDunnett's test to determine where statistically significant differencesexisted between vehicle and drug treated groups. Differences wereconsidered statistically significant at P<0.05. Data analysis wascarried out with GraphPad software (GraphPad Prism).

Results:

An acute administration of the compound of Example 41 (1 and 10 mg/kg,s.c.) to female db/db mice significantly decreased glucose excursion inresponse to an oral glucose challenge (FIG. 24).

Chronic db/db Mouse Studies

Female db/db mice (C57BL/KsJ-Lep^(db/db), Jackson Laboratories, USA)were at 6 weeks of age when received. The mice were housed in atemperature and humidity controlled environment with a 12 h light: 12 hdark cycle, and given access to food (Purina rodent chow 5008) and waterad libitum. The mice (9 week old) were pre-bled 4 days prior to drugtreatment, and those within a narrow range of fasted blood glucoselevels were selected for the study in order to minimize variabilitybetween vehicle control and drug-treated groups. The mice were dosedwith vehicle (s.c.) or the compound of Example 41 (1, 3 and 10 mg/kg,s.c.) once every two days for 3 weeks (N=10 mice/group). Basal fasting(2 to 6 hr) blood glucose measurements were conducted weekly. On studyday 20, an oral glucose tolerance test was performed following a 6 hrfast. Blood samples were collected from tail clips for determination ofbaseline values (t=0 min). The mice were then gavaged with an oral bolusof glucose (1 g/kg), and additional blood samples were collected atregular intervals (t=30, 60, and 120 min) for glucose measurement. Toanalyze the effects of the compound of Example 41 on oral glucosetolerance the absolute difference in blood glucose from baseline(fasting blood glucose, t=0 min) was calculated for each time point. Thearea under the curve (AUC₀₋₁₂₀ min) was determined using the trapezoidmethod.

Data Analysis:

All data shown are the mean±standard deviation (s.d.). Statisticalevaluation of the data was carried out using one-way ANOVA, followed byDunnett's test to determine where statistically significant differencesexisted between vehicle and drug treated groups. Differences wereconsidered statistically significant at P<0.05. Data analysis wascarried out with GraphPad software (GraphPad Prism).

Results:

Chronic administration of the compound of Example 41 (1, 3 and 10 mg/kg,q48 hr, s.c.) to female db/db mice reduced basal blood glucose levels(day 8, 15 and 21) versus vehicle-treated animals during the 3-weektreatment period (FIG. 25A). As shown in FIG. 25B, on day 20 thecompound of Example 41 (both at 3 and 10 mg/kg, q48 hr, s.c.)significantly decreased glucose excursion in response to an oral glucosechallenge.

It is to be understood that the invention is not limited to theparticular embodiments of the invention described above, as variationsof the particular embodiments may be made and still fall within thescope of the appended claims.

1. A neuropeptide-2 receptor agonist of the formula (I):

wherein: X is 4-oxo-6-(1-piperazinyl)-3(4H)-quinazoline-acetic acid(Pqa), Y is H, an acyl moiety, a substituted or unsubstituted alkyl, asubstituted or unsubstituted lower alkyl, a substituted or unsubstitutedaryl, a substituted or unsubstituted heteroaryl, a substituted orunsubstituted alkoxy, a poly(ethylene) glycol moiety, PEG_(m)-SSA,PEG_(m)β-SBA, PEG_(m)-SPA or PEG_(m)-BTC, Y′ is H, a poly(ethylene)glycol moiety, PEG_(m)-SSA, PEG_(m)-β-SBA, PEG_(m)-SPA or PEG_(m)-BTC,R₁ is Ile, Ala, (D) Ile, N-methyl Ile, Aib, 1-1Aic, 2-2 Aic, Ach or Acp,R₂ is Lys, Ala, (D) Lys, NMelys, Nle or (Lys-Gly), R₃ is Arg, Ala,(D)Arg, N-methyl Arg, Phe, 3,4,5-Trifluoro Phe or 2,3,4,5,6-PentafluoroPhe, R₄ is His, Ala, (D)His, N-methyl His, 4-MeOApc, 3-Pal or 4-Pal, R₅is Tyr, Ala, (D) Tyr, N-methyl Tyr, Trp, Tic, Bip, Dip, (1)Nal, (2)Nal,3,4,5-TrifluroPhe or 2,3,4,5,6-Pentafluoro Phe, R₆ is Leu, Ala, (D)Leuor N-methyl Leu, R₇ is Asn, Ala or (D)Asn, R₈ is Leu or Trp, R₉ is Val,Ala, (D) Val or N-methyl Val, R₁₀ is Thr, Ala or N-methyl Thr, R₁₁ isArg, (D) Arg or N-methyl Arg, R₁₂ is Gln or Ala, R₁₃ is Arg, (D)Arg orN-methyl Arg, R₁₄ is Tyr, (D) Tyr or N-methyl Tyr, modified-Tyr, Phe,modified-Phe, (1) Nal, (2) Nal, Cha, C-alpha-methyl Tyr, or Trp, andPEG_(m) is greater than about 1 KDa, or a pharmaceutically acceptablesalt thereof.
 2. The neuropeptide-2 receptor agonist according to claim1, wherein: Y is H or an acyl moiety, and Y′ is a poly(ethylene) glycolmoiety, PEG_(m)-SSA, PEG_(m)-β-SBA, PEG_(m)-SPA or PEG_(m)-BTC.
 3. Theneuropeptide-2 receptor agonist according to claim 1, wherein Y is anacyl moiety.
 4. The neuropeptide-2 receptor agonist according to claim1, wherein said agonist is Ac-IK-Pqa-RHYLNWVTRQ(N-methyl)R (2-6 di F)Y.5. The neuropeptide-2 receptor agonist according to claim 1, whereinsaid agonist is Ac-IK-Pqa-RHYLNWVTRQ(N-methyl)RY.
 6. The neuropeptide-2receptor agonist according to claim 1, wherein said agonist is (PEG30,000)-SPA-IK-Pqa-RHYLNWVTRQ(N-methyl)RY.
 7. The neuropeptide-2receptor agonist according to claim 1, wherein said agonist is(PEG30,000)-SSA-INle-Pqa-RHYLNWVTRQ(N-methyl)RY.
 8. The neuropeptide-2receptor agonist according to claim 1, wherein said agonist isAc-Ile-Lys(PEG30,000 SSA)-Pqa-RHYLNWVTRQ(N-methyl)RY.
 9. Theneuropeptide-2 receptor agonist according to claim 1, wherein saidagonist is H-Ile-Lys(PEG30,000 SSA)-Pqa-RHYLNWVTRQ(N-methyl)RY.
 10. Apharmaceutical composition, comprising a therapeutically effectiveamount of a neuropeptide-2 agonist of the formula (I):

wherein: X is 4-oxo-6-(1-piperazinyl)-3(4H)-quinazoline-acetic acid(Pqa), Y is H, an acyl moiety, a substituted or unsubstituted alkyl, asubstituted or unsubstituted lower alkyl, a substituted or unsubstitutedaryl, a substituted or unsubstituted heteroaryl, a substituted orunsubstituted alkoxy, a poly(ethylene) glycol moiety, PEG_(m)-SSA,PEG_(m)-β-SBA, PEG_(m)-SPA or PEG_(m)-BTC, Y′ is H, a poly(ethylene)glycol moiety, PEG_(m)-SSA, PEG_(m)-β-SBA, PEG_(m)-SPA or PEG_(m)-BTC,R¹ is Ile, Ala, (D) Ile, N-methyl Ile, Aib, 1-1Aic, 2-2 Aic, Ach or Acp,R₂ is Lys, Ala, (D) Lys, NMelys, Nle or (Lys-Gly), R₃ is Arg, Ala,(D)Arg, N-methyl Arg, Phe, 3,4,5-Trifluoro Phe or 2,3,4,5,6-PentafluoroPhe, R₄ is His, Ala, (D)His, N-methyl His, 4-MeOApc, 3-Pal or 4-Pal, R₅is Tyr, Ala, (D) Tyr, N-methyl Tyr, Trp, Tic, Bip, Dip, (1)Nal, (2)Nal,3,4,5-TrifluroPhe or 2,3,4,5,6-Pentafluoro Phe, R₆ is Leu, Ala, (D)Leuor N-methyl Leu, R₇ is Asn, Ala or (D)Asn, R₈ is Leu or Trp, R₉ is Val,Ala, (D) Val or N-methyl Val, R₁₀ is Thr, Ala or N-methyl Thr, R₁₁ isArg, (D) Arg or N-methyl Arg, R₁₂ is Gln or Ala, R₁₃ is Arg, (D)Arg orN-methyl Arg, R₁₄ is Tyr, (D) Tyr or N-methyl Tyr, modified-Tyr, Phe,modified-Phe, (1) Nal, (2) Nal, Cha, C-alpha-methyl Tyr, or Trp, andPEG_(m) is greater than about 1 KDa, or a pharmaceutically acceptablesalt thereof, and a pharmaceutically acceptable carrier.
 11. Thepharmaceutical composition according to claim 10, wherein: Y is H or anacyl moiety, and Y′ is a poly(ethylene) glycol moiety, PEG_(m)-SSA,PEG_(m)-β-SBA, PEG_(m)-SPA or PEG_(m)-BTC.
 12. The pharmaceuticalcomposition according to claim 10, wherein Y is an acyl moiety.
 13. Thepharmaceutical composition according to claim 10, wherein saidneuropeptide-2 agonist is H-Ile-Lys(PEG30,000SSA)-Pqa-RHYLNWVTRQ(N-methyl)RY.
 14. A method of treating a metabolicdisease or disorder, comprising administering to a patient in need ofsaid treatment a therapeutically effective amount of a neuropeptide-2receptor agonist of the formula (I):

wherein: X is 4-oxo-6-(1-piperazinyl)-3(4H)-quinazoline-acetic acid(Pqa), Y is H, an acyl moiety, a substituted or unsubstituted alkyl, asubstituted or unsubstituted lower alkyl, a substituted or unsubstitutedaryl, a substituted or unsubstituted heteroaryl, a substituted orunsubstituted alkoxy, a poly(ethylene) glycol moiety, PEG_(m)-SSA,PEG_(m)-β-SBA, PEG_(m)-SPA or PEG_(m)-BTC, Y′ is H, a poly(ethylene)glycol moiety, PEG_(m)-SSA, PEG_(m)-β-SBA, PEG_(m)-SPA or PEG_(m)-BTC,R₁ is Ile, Ala, (D) Ile, N-methyl Ile, Aib, 1-1Aic, 2-2 Aic, Ach or Acp,R₂ is Lys, Ala, (D) Lys, NMelys, Nle or (Lys-Gly), R₃ is Arg, Ala,(D)Arg, N-methyl Arg, Phe, 3,4,5-Trifluoro Phe or 2,3,4,5,6-PentafluoroPhe, R₄ is His, Ala, (D)His, N-methyl His, 4-MeOApc, 3-Pal or 4-Pal, R₅is Tyr, Ala, (D) Tyr, N-methyl Tyr, Trp, Tic, Bip, Dip, (1)Nal, (2)Nal,3,4,5-TrifluroPhe or 2,3,4,5,6-Pentafluoro Phe, R₆ is Leu, Ala, (D)Leuor N-methyl Leu, R₇ is Asn, Ala or (D)Asn, R₈ is Leu or Trp, R₉ is Val,Ala, (D) Val or N-methyl Val, R₁₀ is Thr, Ala or N-methyl Thr, R₁₁ isArg, (D) Arg or N-methyl Arg, R₁₂ is Gln or Ala, R₁₃ is Arg, (D)Arg orN-methyl Arg, R₁₄ is Tyr, (D) Tyr or N-methyl Tyr, modified-Tyr, Phe,modified-Phe, (1) Nal, (2) Nal, Cha, C-alpha-methyl Tyr, or Trp, andPEG_(m) is greater than about 1 KDa, or a pharmaceutically acceptablesalt thereof.
 15. The method according to claim 14, wherein: Y is H oran acyl moiety, and Y′ is a poly(ethylene) glycol moiety, PEG_(m)-SSA,PEG_(m)-β-SBA, PEG_(m)-SPA or PEG_(m)-BTC.
 16. The method according toclaim 14, wherein said metabolic disease or disorder is obesity, type 2diabetes, metabolic syndrome, insulin resistance, dyslipidemia, impairedfasting glucose and impaired glucose tolerance.
 17. The method accordingto claim 14, wherein said neuropeptide-2 receptor agonist isadministered to said patient once a day.
 18. The method according toclaim 14, wherein said neuropeptide-2 receptor agonist is administeredto said patient once every three days.
 19. The method according to claim14, wherein said neuropeptide-2 receptor agonist is administered to saidpatient once a week.
 20. The method according to claim 14, wherein saidneuropeptide-2 receptor agonist is administered to said patient orally,intranasally, intravenously, subcutaneously, parenterally,transdermally, intraperitoneally, rectally or by inhalation.
 21. Themethod according to claim 14, wherein said neuropeptide-2 receptoragonist is administered intranasally.
 22. The method according to claim14, wherein said neuropeptide-2 receptor agonist is administeredsubcutaneously.
 23. The method according to claim 14, wherein saidneuropeptide-2 receptor agonist is administered at a dosage of fromabout 0.001 to about 100 mg.